cathomycin
naturalproductmech:mibig-d4379ab45b MINTED SEEDED
Grounding review: No 3-star ChEBI entry shares this structure, so the record keeps a content-hashed identifier.
What these status tags mean
Curation status describes a workflow state. SEEDED means the record entered through the source-seeding workflow; it does not imply that no later curation occurred. PROPOSED marks an addition awaiting review; REVIEWED records curator sign-off on identity, structure, filing class and producer claims; DEPRECATED retains a superseded or out-of-scope record. These are workflow decisions, not independently verified human review. Agent-assisted contributions may appear in the record history.
Grounding describes the identifier mapping: EXACT uses an ontology identity asserted by the source; MINTED keeps a local identifier when no defensible ontology identity is available; REVIEW_NEEDED flags an exact structure whose ontology identity needs adjudication. The grounding note retains any unresolved alternatives. These tags do not establish the strength of producer or activity evidence.
Structure
| Standard InChIKey | YJQPYGGHQPGBLI-HHJGLNSVSA-N |
|---|---|
| SMILES | CO[C@@H]1[C@@H](OC(N)=O)[C@@H](O)C(OC2=CC=C3C(O)=C(NC(=O)C4=CC(CC=C(C)C)=C(O)C=C4)C(=O)OC3=C2C)OC1(C)C |
| Stereochemistry | undefined stereocentres — the InChIKey does not distinguish this compound from its stereoisomers |
| Cross-references | npatlas:NPA017352 |
Filing pathway
Shikimates and phenylpropanoids — computed by NPClassifier npclassifier.gnps2.org@2026-09-07, which is why it is pinned rather than recomputed. The gene cluster's asserted class is saccharide, other, from MIBiG.
Producer organisms 1
A claim that this taxon makes the compound. The basis says what the evidence addressed — a knockout and a database assertion are not the same claim.
| Taxon | Basis | Cluster | Evidence |
|---|---|---|---|
| Streptomyces niveus NCBITaxon:193462 |
BGC_CHARACTERIZED | mibig:BGC0000834 | PMID:10770754 |
Occurrences 0
None cited.
Biosynthetic gene clusters 1
| Accession | Host | Locus evidence | Genome |
|---|---|---|---|
| mibig:BGC0000834 | Streptomyces niveus NCBITaxon:193462 |
CLUSTER_DEMONSTRATED | genbank:AF170880.4 |
A locus claim and a taxon claim are different questions. Heterologous expression settles the first and leaves the second where the isolation report left it.
Mechanism & Biosynthesis Overview
Causal graph — biosynthesis 17 nodes, 17 edges
Deposited NovL identity and bounded novobiocic-acid precursor ligation (review #550). The MIBiG BGC0000834 cathomycin owner has one unspecified sugar stereocentre: its InChI /t23-,25+,26-,29? differs from the fully specified novobiocin CID 54675769 /t23-,25+,26-,29-. Their exact InChIKeys differ. Unglycosylated precursor assays neither resolve this centre nor establish synthesis of mature cathomycin/novobiocin. Q9L9F6 denotes the deposited native sequence, not a tagged preparation or a retrospectively identified historical sample. Galm 2003 direct experiments are distinct from the abstract-only 2000 paper. Galm PDF pages exceed printed pages by four; Metzger 2010 PDF pages by twelve.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novL locus from NCIMB 9219 | The owner is the MIBiG v5 cathomycin structure concept, not an exact-key merge with fully specified novobiocin. Its deposited cluster contains novL at 12509..14092(+). | https://mibig.secondarymetabolites.org/go/BGC0000834.5 |
| Deposited novL locus from NCIMB 9219 | encodes_native_protein | Deposited native NovL ligase | AF170880.4/AAF67505.1 has a 1584-nt CDS including stop. CDS-aware table-11 translation gives 527 aa exactly matching Q9L9F6 and the complete printed NovL alignment in Metzger Figure 3.27. Current GenBank source is S. niveus NCIMB 9219. Flinspach et al. retested DSM 40088 (=9219) and DSM 40292 (=11891), then corrected the historical AF170880 strain attribution to 9219. The strains are not synonyms. The 2000 abstract names S. spheroides NCIMB 11891; this historical label does not uniquely establish the internal sequence or provenance of each recombinant sample. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Galm pMS80-derived C-terminal His6 NovL preparation | was_constructed_from_novl | Deposited novL locus from NCIMB 9219 | Galm reused pMS80, a reported 5.0-kb pQE70 construct. Table 13 describes a 1.6-kb SphI/BamHI insert, whereas the results say the existing pMS80/pMS90 constructs were cloned in the same manner as the described SphI/BglII CloL construct. Preserve this source difference; no pMS80 junction sequence or primer-derived A2P substitution is asserted from the CloL/CouL constructs. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| E. coli XL1 Blue MRF' expression host | expressed | Galm pMS80-derived C-terminal His6 NovL preparation | The source used 100 mL LB with 50 ug/mL carbenicillin, 30 C, 180 rpm, OD600 0.7-0.8 at induction and 0.5 mM IPTG for three hours. These are heterologous protein-preparation conditions, not native antibiotic production measurements. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
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| Galm pMS80-derived C-terminal His6 NovL preparation | has_purification_readout | NovL induction and Ni-NTA purification readout | Ni-NTA purification followed by Sephadex G-25 buffer exchange yielded the preparation. Figure 12 NovL lanes 1-5 show marker, pre-induction, post-induction, soluble protein and eluate. Near homogeneity is the source description, not a measured purity percentage or a native assembly determination. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
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| Galm pMS80-derived C-terminal His6 NovL preparation | catalyzes | Galm NovL ligation assay with methylated ring B | Galm assays used 100 uL, 1 mM acid, 1 mM C-8-methylated ring B, 5 mM ATP, 5 mM MnCl2, 50 mM Tris-HCl pH 8.0 and 0.5-2.0 ug enzyme for 30 min at 30 C, stopped with 5 uL 1.5 M trichloroacetic acid. UniProt RHEA:36699 describes unmethylated ring B and 8-desmethylnovobiocic acid; that reaction identifier is deliberately not assigned to this assay. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| 3-Dimethylallyl-4-hydroxybenzoic acid (DMAHB) | is_acyl_substrate_for | Galm NovL ligation assay with methylated ring B | DMAHB is the genuine acid substrate used for the NovL specific-activity measurement and its 100% panel reference. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| 3-Amino-4,7-dihydroxy-8-methylcoumarin | is_amino_substrate_for | Galm NovL ligation assay with methylated ring B | The supplied aminocoumarin bears the C-8 methyl group. This in-vitro acceptance does not by itself establish the order of methylation and ligation in the native producer. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
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| Galm NovL ligation assay with methylated ring B | forms | Assay-assigned novobiocic acid | Products were extracted twice with ethyl acetate and analyzed by HPLC at 305 nm. Quantitation used a novobiocic-acid standard calibration. The product is an unglycosylated precursor, not mature cathomycin/novobiocin. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
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| Galm NovL ligation assay with methylated ring B | has_reported_specific_activity | NovL specific activity in the Galm preparation | The preparation gave 14.5 nkat/mg protein with DMAHB and methylated ring B. Protein was quantified by Bradford with BSA calibration. The source does not provide a replicate count or uncertainty for this value; it is not kcat or native flux. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
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| Heat-inactivated NovL assay comparator | is_reported_control_for | Galm NovL ligation assay with methylated ring B | Methods specify protein heated for 30 min at 100 C with all other components present. This records the control design, not an independently quantified zero-product measurement. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
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| Galm pMS80-derived C-terminal His6 NovL preparation | was_tested_in | Galm NovL aromatic-acid substrate panel | The HPLC panel compares the genuine acids and 22 synthetic ring-A analogues. NovL and CloL normalize to DMAHB, but CouL normalizes to its pyrrole dicarboxylic acid. Relative activities therefore do not share an absolute cross-enzyme denominator. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
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| Galm NovL aromatic-acid substrate panel | supports_bounded_substrate_scope | Condition-specific acceptance of selected acid analogues | NovL weakly accepts 4-hydroxybenzoic acid and accepts the 3-chloro/3-bromo analogues; the displayed assay does not accept the pyrrole dicarboxylic acid, the cyclohexyl analogue or 3,5-dibromo-4-hydroxybenzoic acid. These negatives apply to this preparation and assay, not every enzyme loading. Figure 13 shows no NovL bar for RAA380, whereas the 2000 abstract reports low acceptance of 3-methyl-4-aminobenzoate. Preserve that difference without inventing a reconciled detection limit. LC-MS/MS supports the panel products collectively; it is not a separate enzyme-specific structural proof for every signal. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| His6 NovL preparation reported in the 2000 paper | was_reported_as_novl_product | Deposited novL locus from NCIMB 9219 | The 2000 abstract reports His6 NovL expressed in E. coli and purified by affinity and gel chromatography. Full primary methods were not obtained; do not transfer Galm host, buffer or construct-junction details to this preparation. | DOI:10.1074/jbc.M003066200
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| His6 NovL preparation reported in the 2000 paper | catalyzes_reported_ligation | Assay-assigned novobiocic acid | The primary abstract reports ATP-dependent amide formation from DMAHB and 3-amino-4,7-dihydroxy-8-methylcoumarin. | DOI:10.1074/jbc.M003066200
|
| His6 NovL preparation reported in the 2000 paper | shows_reported_activation | Reported ATP-PPi exchange and acyl activation | ATP-PPi exchange supports the reported acyl-adenylate activation step. This is not a directly visualized catalytic intermediate or an independently measured native-cell flux. | DOI:10.1074/jbc.M003066200
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| His6 NovL preparation reported in the 2000 paper | has_reported_biochemical_properties | Abstract-reported NovL biochemical properties | The abstract reports a gel-filtration-active monomer, ATP specificity, Mg2+ or Mn2+ dependence and apparent Km values of 19 uM for DMAHB and 131 uM for methylated ring B. These are source-reported values; their detailed assay and uncertainty were not independently recovered. | DOI:10.1074/jbc.M003066200
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 18 nodes, 18 edges
Metzger 2010 NovL preparations, precursor activity and unsuccessful structure determination (review #550). Native Q9L9F6 is a sequence reference, not the identity of the His8 fusion, thrombin-cleaved scar protein, SeMet derivative or chemically methylated sample. Specific activities refer to methylated-ring-B precursor assays, not mature product synthesis. A diffraction dataset without solved phases does not establish residue contacts or biological oligomer stoichiometry. All page numbers below are PDF pages, twelve higher than printed pages. The later NovL/CouL chimera study is a separate lead.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited native NovL sequence reference | is_native_sequence_reference_for | pUM07 NovL expression construct | The source amplified novL from nov-BG1 into pGEM-T (pUM03), then pHis8 (pUM07); it reports sequence verification. The full printed 527-aa NovL sequence matches deposited AAF67505.1 and Q9L9F6 exactly. The current deposit identifies NCIMB 9219, following the historical strain correction. No complete experimental vector sequence was independently inspected. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| pUM07 NovL expression construct | encodes_engineered_fusion | pUM07 N-terminal His8 NovL fusion | Literal novLHis8_f TGGGATCCAACAAGGATCACGCACC and novLHis8_r TCGAATTCCCCACCTCACCTGTCCA reconstruct a 1600-nt PCR product and 1590-nt BamHI/EcoRI restriction fragment, as reported. Their unique 17-nt 3-prime matches extrapolate to 12507..12531(+) and 14106..14082(-). The model retains native residues 3-527 and the stop, replacing the native N terminus with vector-derived sequence. No native accession is assigned to this engineered protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| E. coli BL21(DE3)pLysS expression host | expressed | pUM07 N-terminal His8 NovL fusion | The described preparation used 1 L TB medium, induction at OD600 0.6 with 0.5 mM IPTG after cooling to 20 C, and six hours at 250 rpm. IMAC preceded further purification. A separate retained-His-tag preparation is reported at 40 mg/L culture and final 18 mg/mL, with a 59-kDa SDS-PAGE assignment; those values are not the cleaved-protein concentration. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| pUM07 N-terminal His8 NovL fusion | was_thrombin_processed_to | Thrombin-cleaved NovL with reported GSHGGS scar | Thrombin treatment (0.1 U/mg, 15 h at 4 C), removal of tag and thrombin, and Superdex 200 purification yielded the crystallization preparation. The reported post-cleavage N terminus is GSHGGSNK..., with GSHGGS replacing native MA; cleavage therefore does not restore native Q9L9F6. Figure 3.22 shows induction, soluble fraction, affinity eluate, cleavage and final material. Table 3.5 gives 40 mg/L culture and final 19.5 mg/mL, not assay loading or antibiotic titer. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| pUM07 NovL expression construct | was_used_for | NovL SeMet preparation for crystallization | The same pUM07 construct supplied the SeMet preparation; this is incorporation during heterologous expression, not a native NovL isoform or a genetically encoded methionine substitution. The source gives 15 mg/L culture and final 19 mg/mL after the crystallization-purification workflow. It does not quantify site-by-site SeMet occupancy. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| E. coli B834(DE3)pLysS SeMet expression host | expressed | NovL SeMet preparation for crystallization | B834(DE3)pLysS was grown in SeMet medium after washing the methionine-grown inoculum; production medium contained 0.04 mg/mL L-selenomethionine. Methods specify 1 mM IPTG after cooling to 20 C, distinct from the ordinary expression protocol. SeMet occupancy and full sample sequence were not independently measured here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Thrombin-cleaved NovL with reported GSHGGS scar | catalyzes | Metzger NovL precursor-ligation assay | Methods use 100 uL with 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM ATP and 1 mM of each substrate, incubated 15 min at 30 C before acid quench. Enzyme loading for the Table 3.5 value is not specified separately; do not borrow the later chimera study loading or Galm Mn2+/5-mM-ATP protocol. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| 3-Dimethylallyl-4-hydroxybenzoic acid | is_acyl_substrate_for | Metzger NovL precursor-ligation assay | DMAHB is explicitly the acid used for the reported NovL and NovL-SeMet activity measurements. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| 3-Amino-4,7-dihydroxy-8-methylcoumarin | is_amino_substrate_for | Metzger NovL precursor-ligation assay | The methods identify the C-8-methylated aminocoumarin. No unmethylated-ring-B reaction identifier or in-vivo methylation order is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Metzger NovL precursor-ligation assay | forms | HPLC-assigned novobiocic acid | Ethyl-acetate extraction and HPLC at 330 nm with a 1-mM novobiocic-acid standard support the source-assigned product. This is not an assay of glycosylation or mature antibiotic synthesis. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Thrombin-cleaved NovL with reported GSHGGS scar | has_reported_specific_activity | Specific activity of the cleaved NovL preparation | Table 3.5 reports 13.0 nkat/mg with DMAHB. Purified-protein concentration methods use A280 and sequence-derived extinction coefficients; the NovL table gives 0.75 with tag and 0.77 without tag in L/g/cm. No uncertainty or replicate count accompanies this activity. Difference from Galm 14.5 nkat/mg is not a controlled test of tag position or cleavage effects. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| NovL SeMet preparation for crystallization | catalyzes | Metzger NovL precursor-ligation assay | The source directly reports an activity assay with DMAHB and subsequent HPLC for the SeMet preparation; activity is not inferred solely from the SDS-PAGE band. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| NovL SeMet preparation for crystallization | has_reported_specific_activity | Specific activity of the NovL SeMet preparation | The source reports 8.4 nkat/mg. This preparation-level measurement, without stated uncertainty, does not establish the effect of each SeMet replacement on kcat or binding. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Thrombin-cleaved NovL with reported GSHGGS scar | formed | NovL crystals from the initial screen | An initial screen of 2712 conditions at 4 C found a NovL condition; crystals reached about 100 x 100 x 50 um after two weeks. Many were twinned. Heavy-metal soaking and co-crystallization trials did not solve the phase problem; added ATP/analogues/substrates are not observed bound ligands. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| NovL SeMet preparation for crystallization | formed | Twinned NovL SeMet crystals | The SeMet preparation formed approximately 50 x 50 x 20 um crystals under similar conditions, but at least 20% twinning prevented their use for phase determination. This is not a solved SeMet structure or evidence of a biological complex. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| NovL crystals from the initial screen | had_reported_diffraction_follow_up | Reported unphased 2.6-A NovL diffraction dataset | The thesis reports a dataset from one untwinned NovL crystal to 2.6 A at ESRF ID14-2, but explicitly says the structure was not solved. The reported two molecules per asymmetric unit do not establish a native dimer. The dataset paragraph does not uniquely identify a sequenced preparation or a ligand-bound state; no PDB accession is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Thrombin-cleaved NovL with reported GSHGGS scar | was_chemically_modified_for_crystallization | Chemically methylated NovL preparation | Methods describe reductive methylation with DMAB/formaldehyde and subsequent size-exclusion purification, intended to modify lysines and the N terminus. The NovL result reports 19% protein recovery relative to input and final 14 mg/mL. Recovery is not methylation occupancy or residual enzyme activity; site-specific modification was not established. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Chemically methylated NovL preparation | formed | Small crystals of chemically methylated NovL | One tested condition yielded small crystals still being optimized at thesis completion. No solved structure or activity value is supplied for this chemically modified preparation. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 20 nodes, 28 edges
Ten engineered NovL/CouL amide-synthetase chimeras from Metzger 2010 (review #551), associated with the NovL precursor mechanism of the cathomycin/BGC0000834 owner. These are experimental fusion proteins, not ten native UniProt entries or ten natural-product structures. Parent sequence references, schematic swaps, literal-primer models and actual assayed preparations are distinct. The thesis reports sequence verification, but complete experimental plasmids were not independently obtained. Figure 3.27 CouL does not exactly match any of AF235050.1-.4. All page locators below are PDF pages, twelve above printed pages.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited native NovL sequence reference | is_sequence_reference_for | pUM07 NovL parental DNA construct | All 527 NovL residues printed in Figure 3.27 match the CDS-aware AF170880.4/AAF67505.1 translation and Q9L9F6. pUM07 is an engineered N-terminal His-tag construct, not native Q9L9F6. Current AF170880.4 is attributed to NCIMB 9219 following the published strain correction; historical strain labels do not uniquely identify each assay sample. No matched parent batch or tag-cleavage state is supplied in the chimera activity table. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Current deposited native CouL sequence reference | is_sequence_reference_for | pUM05 CouL parental DNA construct | Current deposited CouL (UniProtKB:Q9F8U8, AF235050.4:10634..12223(+), AAG29784.2, 529 aa, sequence version 2) has Glu-Gly at residues 384-385, whereas historical AF235050.1-.3 / AAG29784.1 had Arg-Ala and printed Figure 3.27 displays Glu-Ala. pUM05 (cou_parent) is an N-terminally His-tagged pET28a CouL parental construct and is therefore not identical to the untagged native Q9F8U8 sequence. | https://www.uniprot.org/uniprotkb/Q9F8U8 |
| Printed CouL parent and deposit-version discrepancy | limits_exact_identity_of | pUM05 CouL parental DNA construct | Figure 3.27 prints a 529-aa CouL with EA384-385. AF235050.1-.3/AAG29784.1 have RA, whereas AF235050.4/AAG29784.2 has EG. All other printed residues agree. Thus neither historical nor current deposit exactly identifies the printed parent. A printing error or sample difference is unresolved; this is not proof of a third biological allele. pUM05 was derived from couMW12 via pUM10. The table reports a 1607-bp PCR product and a 1615-bp BspHI/NotI subclone; without the full vector sequence, these do not establish exact junctions or an impossible cloning event. CouL-derived segments in Chim1-4 and Chim12 include positions 384-385. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| pUM07 NovL parental DNA construct | supplied_template_segments_to | Four-stage fusion-PCR and pHis8 construct panel | Table 2.5 identifies pUM07 as NovL template. Region I is 161-171; IIa is 192-214; IIb is 215-229; the C-terminal region starts at 413. These source-defined regions guide the schematic swaps, not uniquely sequenced crossover coordinates. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| pUM05 CouL parental DNA construct | supplied_template_segments_to | Four-stage fusion-PCR and pHis8 construct panel | Table 2.5 identifies pUM05 as CouL template. Region I is 161-171; IIa is 192-216; IIb is 217-231; the C-terminal region starts at 415. The source informally calls the C terminus the last 100 residues, but 415-529 comprises 115 residues. No exact 100-residue construct boundary is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Four-stage fusion-PCR and pHis8 construct panel | has_literal_primer_audit | Literal-primer reconstruction and schematic-boundary limits | All 15 printed primer strings were checked; each of the six internal forward/reverse pairs is reverse-complementary. Full-length ungapped matches within the deposited parent CDSs uniquely localize the internal primers despite template mismatches; short exact suffixes alone can select unrelated genomic positions. The shared reverse primer GAATGAAGCTTCTCACCTGTCCACCAGGACGTC differs from CouL six bases from its 3-prime end, reconstructing synonymous GTG-to-GTC at native CouL V525. The models use NovL AF170880.4 and historical CouL AF235050.3, not a recovered pUM05 sequence. Relative to simple concatenation at the stated region boundaries, Chim4 retains NovL R241/A245 in place of CouL K243/T247; Chim8 retains NovL S154/V155 in place of CouL T154/A155. The other eight modeled internal cores match that simple combination. Conserved sequence can make a crossover nonunique. These are inferred models excluding native residues 1-2 and unknown vector-derived sequence, not experimental sequence determinations. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| E. coli BL21(DE3)pLysS expression host | supplied_expression_material_for | Chimera IMAC and PD-10 preparation workflow | Expression used 1 L TB culture, cooling at OD600 0.6, 0.5 mM IPTG and six hours at 20 C/250 rpm. Proteins were expressed with an N-terminal His tag and purified by IMAC. For chimeras, pooled fractions underwent PD-10 desalting, addition of 15% glycerol and storage at -70 C. The separate thrombin/size-exclusion protocol for crystallization proteins does not establish tag removal for these chimeras. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Chimera IMAC and PD-10 preparation workflow | has_reported_readout | Reported chimera SDS-PAGE purification readout | Figure 3.28 displays all ten chimeras at 20 ug per lane and labels them 57 kDa, but their apparent migrations differ. Near purity is the author description, not a quantified purity percentage, folding/stability control or biological oligomer determination. Table 3.7 gives dashes for both parental yield/concentration rows; earlier NovL preparations must not be substituted as matched measurements. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim1 NovL/CouL His-tagged fusion (pUM27) | Chim1: NovL-derived N-terminal segment and CouL-derived C-terminal segment, schematic transition before region I. Table 2.5 uses NovCouMut2f/r for the internal overlap. pGEM-T intermediate pUM18 precedes pHis8 construct pUM27. The reported 1608-bp PCR and 1592-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim1 NovL/CouL His-tagged fusion (pUM27) | Table 3.7 reports 105 mg recovered protein per L culture and final concentration 1.0 mg/mL for pUM27. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim2 NovL/CouL His-tagged fusion (pSF06) | Chim2: NovL-derived N-terminal segment and CouL-derived C-terminal segment, schematic transition after region I. Table 2.5 uses NovCouMut1f/r for the internal overlap. pGEM-T intermediate pSF01 precedes pHis8 construct pSF06. The reported 1608-bp PCR and 1592-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim2 NovL/CouL His-tagged fusion (pSF06) | Table 3.7 reports 127 mg recovered protein per L culture and final concentration 4.1 mg/mL for pSF06. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim3 NovL/CouL His-tagged fusion (pSF09) | Chim3: NovL-derived N-terminal segment and CouL-derived C-terminal segment, schematic transition after region IIa. Table 2.5 uses NovCouMut4af/r for the internal overlap. pGEM-T intermediate pSF08 precedes pHis8 construct pSF09. The reported 1602-bp PCR and 1586-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim3 NovL/CouL His-tagged fusion (pSF09) | Table 3.7 reports 90 mg recovered protein per L culture and final concentration 4.0 mg/mL for pSF09. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim4 NovL/CouL His-tagged fusion (pUM24) | Chim4: NovL-derived N-terminal segment and CouL-derived C-terminal segment, schematic transition after region IIb. Table 2.5 uses NovCouMut3f/r for the internal overlap. pGEM-T intermediate pUM20 precedes pHis8 construct pUM24. The reported 1602-bp PCR and 1586-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim4 NovL/CouL His-tagged fusion (pUM24) | Table 3.7 reports 82 mg recovered protein per L culture and final concentration 5.6 mg/mL for pUM24. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim5 NovL/CouL His-tagged fusion (pUM43) | Chim5: NovL-derived N-terminal segment and CouL-derived C-terminal segment, schematic transition before the C-terminal region. Table 2.5 uses NovCouMut5f/r for the internal overlap. pGEM-T intermediate pUM45 precedes pHis8 construct pUM43. The reported 1602-bp PCR and 1586-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim5 NovL/CouL His-tagged fusion (pUM43) | Table 3.7 reports 113 mg recovered protein per L culture and final concentration 7.5 mg/mL for pUM43. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim8 NovL/CouL His-tagged fusion (pSF07) | Chim8: CouL-derived N-terminal segment and NovL-derived C-terminal segment, schematic transition before region I. Table 2.5 uses NovCouMut2f/r for the internal overlap. pGEM-T intermediate pSF03 precedes pHis8 construct pSF07. The reported 1602-bp PCR and 1586-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim8 NovL/CouL His-tagged fusion (pSF07) | Table 3.7 reports 50 mg recovered protein per L culture and final concentration 3.4 mg/mL for pSF07. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim9 NovL/CouL His-tagged fusion (pSF04) | Chim9: CouL-derived N-terminal segment and NovL-derived C-terminal segment, schematic transition after region I. Table 2.5 uses NovCouMut1f/r for the internal overlap. pGEM-T intermediate pUM21 precedes pHis8 construct pSF04. The reported 1602-bp PCR and 1586-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim9 NovL/CouL His-tagged fusion (pSF04) | Table 3.7 reports 94 mg recovered protein per L culture and final concentration 5.2 mg/mL for pSF04. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim10 NovL/CouL His-tagged fusion (pUM25) | Chim10: CouL-derived N-terminal segment and NovL-derived C-terminal segment, schematic transition after region IIa. Table 2.5 uses NovCouMut4bf/r for the internal overlap. pGEM-T intermediate pUM22 precedes pHis8 construct pUM25. The reported 1608-bp PCR and 1592-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim10 NovL/CouL His-tagged fusion (pUM25) | Table 3.7 reports 76 mg recovered protein per L culture and final concentration 3.8 mg/mL for pUM25. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim11 NovL/CouL His-tagged fusion (pSF05) | Chim11: CouL-derived N-terminal segment and NovL-derived C-terminal segment, schematic transition after region IIb. Table 2.5 uses NovCouMut3f/r for the internal overlap. pGEM-T intermediate pUM23 precedes pHis8 construct pSF05. The reported 1608-bp PCR and 1592-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim11 NovL/CouL His-tagged fusion (pSF05) | Table 3.7 reports 68 mg recovered protein per L culture and final concentration 3.8 mg/mL for pSF05. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Four-stage fusion-PCR and pHis8 construct panel | generated_expression_construct_for | Chim12 NovL/CouL His-tagged fusion (pUM40) | Chim12: CouL-derived N-terminal segment and NovL-derived C-terminal segment, schematic transition before the C-terminal region. Table 2.5 uses NovCouMut5f/r for the internal overlap. pGEM-T intermediate pUM39 precedes pHis8 construct pUM40. The reported 1608-bp PCR and 1592-bp BspHI/HindIII fragment agree with the literal deposited-template model. This does not resolve parent sequence differences, tag/junction sequence or the independently unobserved full experimental fusion. No native UniProt accession is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| Chimera IMAC and PD-10 preparation workflow | yielded_preparation | Chim12 NovL/CouL His-tagged fusion (pUM40) | Table 3.7 reports 83 mg recovered protein per L culture and final concentration 6.3 mg/mL for pUM40. These values are neither assay load nor natural-product titer. Purified-protein quantification methods describe A280 with sequence-derived extinction coefficients; per-chimera coefficients and measurement uncertainties are not tabulated here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 25 nodes, 24 edges
Metzger 2010 Figure 3.31 endpoint assays of ten NovL/CouL chimeras and two parental comparators (review #551). Each protein has its own reported six-column activity vector, retaining substrate, product, loading, normalization and censoring. Comparisons support bounded regional contributions to precursor-ligation activity, not exact substrate-contact residues, catalytic efficiencies or mature antibiotic synthesis. The cathomycin owner has an unspecified sugar stereocentre; unglycosylated assays do not resolve it or justify an exact-structure merge. Construct identity is qualified in the separate chimera preparation graph. PDF locators are printed pages +12.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| pUM05-derived CouL parental comparator | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [3.3, 0.7, 100, 100, 100, 100]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim1 NovL/CouL His-tagged fusion (pUM27) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [5.8, 1.0, 187, 85, 135, 80]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim2 NovL/CouL His-tagged fusion (pSF06) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [3.5, 0.4, 114, 29, 111, 67]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim3 NovL/CouL His-tagged fusion (pSF09) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [11, 1.5, 41, <NG, 55, 35]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim4 NovL/CouL His-tagged fusion (pUM24) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [7.6, 1.0, 15, <NG, 33, 35]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim5 NovL/CouL His-tagged fusion (pUM43) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [106, 89, <NG, <NG, 3.5, <NG]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| pUM07-derived NovL parental comparator | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [100, 100, <NG, <NG, 3.9, <NG]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. NovL S3 monoamide is below detection at 4 ug but 3.9% of the matched CouL reference at 40 ug; earlier negative assays are not universal substrate rejection. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim8 NovL/CouL His-tagged fusion (pSF07) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [119, 119, <NG, <NG, 6.1, <NG]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim9 NovL/CouL His-tagged fusion (pSF04) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [109, 97, <NG, <NG, 5.3, <NG]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim10 NovL/CouL His-tagged fusion (pUM25) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [82, 48, <NG, <NG, 6.2, <NG]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim11 NovL/CouL His-tagged fusion (pSF05) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [24, 23, <NG, <NG, 8.4, <NG]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Chim12 NovL/CouL His-tagged fusion (pUM40) | has_reported_relative_activity_in | Six-column relative-activity endpoint panel | Reported activity [%], columns in source order: [4.6, 0.9, 85, 82, 106, 89]. Columns 1-2: S1 and S2 with 4 ug enzyme, each relative to NovL with the same substrate (=100%). Columns 3-4: S3 monoamide and diamide with 4 ug; columns 5-6: S3 monoamide and diamide with 40 ug. Each S3 column uses its own load/product-matched CouL reference (=100%). <NG means below detection, not zero; the numerical detection threshold is unstated. These values are not absolute conversion percentages or one shared activity scale. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| S1: 3-dimethylallyl-4-hydroxybenzoic acid | was_substrate_in | Six-column relative-activity endpoint panel | S1 is the genuine NovL acid substrate used for novobiocic-acid formation. Assays used 100 uL, 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM ATP and 1 mM of each supplied substrate, incubated 15 min at 30 C and quenched with 5 uL 1.5 M trichloroacetic acid. Protein load is column-specific, 4 or 40 ug. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| S2: 3-propyl-4-hydroxybenzoic acid | was_substrate_in | Six-column relative-activity endpoint panel | S2 is an acid analogue; activity is a surrogate-substrate result, not native synthesis. Assays used 100 uL, 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM ATP and 1 mM of each supplied substrate, incubated 15 min at 30 C and quenched with 5 uL 1.5 M trichloroacetic acid. Protein load is column-specific, 4 or 40 ug. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| S3: 3-methylpyrrole-2,4-dicarboxylic acid | was_substrate_in | Six-column relative-activity endpoint panel | S3 has two carboxyl groups; the monoamide and diamide readouts are separate. Assays used 100 uL, 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM ATP and 1 mM of each supplied substrate, incubated 15 min at 30 C and quenched with 5 uL 1.5 M trichloroacetic acid. Protein load is column-specific, 4 or 40 ug. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| 3-Amino-4,7-dihydroxy-8-methylcoumarin | was_substrate_in | Six-column relative-activity endpoint panel | The supplied aminocoumarin is C8-methylated. No unmethylated-ring-B Rhea reaction or in-vivo methylation order is assigned. Assays used 100 uL, 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM ATP and 1 mM of each supplied substrate, incubated 15 min at 30 C and quenched with 5 uL 1.5 M trichloroacetic acid. Protein load is column-specific, 4 or 40 ug. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Six-column relative-activity endpoint panel | measured_source_assigned_product | S1-derived novobiocic acid readout | S1 yields the source-assigned unglycosylated novobiocic-acid signal. Ethyl-acetate extraction and C18 HPLC at 330 nm used a 1-mM novobiocic-acid standard. Separate product-specific response factors and independent structural proofs for each chimera signal are not provided in this experiment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Six-column relative-activity endpoint panel | measured_source_assigned_product | S2-derived surrogate amide readout | S2 yields the source-assigned surrogate amide signal. Ethyl-acetate extraction and C18 HPLC at 330 nm used a 1-mM novobiocic-acid standard. Separate product-specific response factors and independent structural proofs for each chimera signal are not provided in this experiment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Six-column relative-activity endpoint panel | measured_source_assigned_product | S3/1 source-assigned monoamide readout | S3/1 denotes monoamide product 1; no unique monoamide regiochemistry is assigned here. Ethyl-acetate extraction and C18 HPLC at 330 nm used a 1-mM novobiocic-acid standard. Separate product-specific response factors and independent structural proofs for each chimera signal are not provided in this experiment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Six-column relative-activity endpoint panel | measured_source_assigned_product | S3/2 source-assigned diamide readout | S3/2 denotes diamide product 2; it is not mature coumermycin A1 or a directly observed catalytic intermediate. Ethyl-acetate extraction and C18 HPLC at 330 nm used a 1-mM novobiocic-acid standard. Separate product-specific response factors and independent structural proofs for each chimera signal are not provided in this experiment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| Six-column relative-activity endpoint panel | is_limited_by | Parent enzyme-loading linearity limits | Figure 3.30 tested parental enzyme loads up to 6 ug per 100 uL. NovL product formation was linear only up to 2 ug for S1 and 3.5 ug for S2. The text describes CouL as linear beyond 6 ug, although the plotted range ends at 6. The selected 4-ug panel load exceeds both stated NovL bounds, and 40-ug S3 results are outside this tested loading range. Figure 3.31 reports duplicate assays without specifying biological replicates or tabulated uncertainty. The Figure 3.30 error bars are not identified here as SD or SE. Thus these endpoint comparisons must not become initial-rate kinetics, kcat or catalytic-efficiency estimates. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Six-column relative-activity endpoint panel | supports_bounded_interpretation | Conditional regional-swap activity comparisons | The selected swaps associate region IIa, region IIb and the following segment with activity differences. For S1, Chim5/8/9 have near-NovL normalized values, while Chim10/11 are lower; for S3 the author emphasizes the 40-ug monoamide column because other signals are weak. This does not establish universal dispensability of the termini or region I, direct substrate binding by particular residues, or loss of folding in less active fusions. Normalized columns cannot be compared as an absolute protein-dose response. Homology-based candidate residues on PDF pp.97-99 remain untested hypotheses in this study; no extra point-mutant proteins or solved NovL/CouL structures are inferred. Below-detection entries are retained as censored observations. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Deposited native NovL sequence reference | is_native_reference_for | pUM07-derived NovL parental comparator | UniProtKB:Q9L9F6 is the deposited native sequence reference for pUM07-derived NovL parental comparator (node 'nov_parent'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F6 |
| Deposited native CouL sequence reference | is_native_reference_for | pUM05-derived CouL parental comparator | UniProtKB:Q9F8U8 is the deposited native sequence reference for pUM05-derived CouL parental comparator (node 'cou_parent'); the experimental preparation node remains review_needed because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9F8U8 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 13 nodes, 12 edges
NovQ native sequence, source-taxonomy conflict and bounded Ozaki 2009 precursor evidence (review #554). The BGC0000834 owner is not merged with fully specified novobiocin. The deposited NCIMB 9219 sequence is grounded; the strain-16259 source and recombinant sample retain identity uncertainty. Only the primary abstract was obtained for biochemical claims.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novQ locus from NCIMB 9219 | The BGC0000834.5 cathomycin owner links AF170880.4, whose novQ CDS is 19093..20064(+). The owner retains its unspecified sugar stereocentre; this locus association is not a new exact-structure equivalence assertion. | https://mibig.secondarymetabolites.org/go/BGC0000834.5 |
| Deposited novQ locus from NCIMB 9219 | encodes_native_protein | Deposited native NovQ sequence reference | AF170880.4/AAF67510.2 has 972 coding nt including stop. CDS-aware table-11 translation exactly matches deposited AAF67510.2 and all 323 residues of Q9L9F1. Current GenBank source is S. niveus NCIMB 9219 (taxon 193462). UniProt reference 1 still lists the historical NCIMB 11891 strain group; that metadata does not override the corrected current nucleotide deposit or identify every historical assay sample. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited native NovQ sequence reference | has_identical_deposited_sequence_to | NovQ deposit from strain 16259 with conflicting taxon labels | AB496950.1/BAI44329.1 novQ is 81..1052(+), also 972 nt including stop and 323 aa. Its CDS-aware translation matches Q9L9F1 and AAF67510.2 exactly. This equality concerns protein sequences, not strain or taxon synonymy and not the complete sequence of a recombinant construct. | https://www.ncbi.nlm.nih.gov/nuccore/AB496950.1 |
| NovQ deposit from strain 16259 with conflicting taxon labels | has_unresolved_source_taxonomy | Strain 16259 source-taxonomy disagreement | The AB496950.1 record dated 2023-01-05 names Actinoalloteichus caeruleus, taxon 2893586, strain 16259, with Streptomyces caeruleus/niveus synonym notes. UniProt uses S. niveus taxon 193462 and links strain 16259 to Ozaki 2009; the primary abstract also names S. niveus. These source labels are preserved without changing the owner producer or asserting that strain 16259 is NCIMB 9219 or NCIMB 11891. | https://www.ncbi.nlm.nih.gov/nuccore/AB496950.1 |
| NovQ deposit from strain 16259 with conflicting taxon labels | is_database_linked_to_study_preparation | Ozaki 2009 recombinant NovQ preparation | UniProt associates the Ozaki study with strain 16259 and AB496950. The full paper and complete experimental construct sequence were not obtained. The deposited native sequence is not assigned as the exact identity of a tag-bearing, processed or otherwise engineered sample. | https://www.uniprot.org/uniprotkb/Q9L9F1 |
| Ozaki E. coli expression host | expressed | Ozaki 2009 recombinant NovQ preparation | The abstract reports E. coli expression and purification to homogeneity. Host strain, fusion junctions and detailed purification conditions are not supplied by the inspected abstract. | DOI:10.1038/ja.2009.48
|
| Ozaki 2009 recombinant NovQ preparation | has_reported_preparation_properties | Reported soluble monomeric recombinant preparation | The source describes soluble monomeric protein of about 40 kDa. This is a recombinant-preparation observation, not an exact native mass or cellular stoichiometry measurement. | DOI:10.1038/ja.2009.48
|
| Ozaki 2009 recombinant NovQ preparation | catalyzes_reported_prenylation | Abstract-reported NovQ precursor prenylation | The abstract reports divalent-cation-independent dimethylallylation. It mentions kinetic analysis but supplies no constants, assay recipe or uncertainty; none is reconstructed here. | DOI:10.1038/ja.2009.48
|
| 4-Hydroxyphenylpyruvate | is_reported_acceptor_for | Abstract-reported NovQ precursor prenylation | 4-HPP is the reported aromatic acceptor, not mature cathomycin. | DOI:10.1038/ja.2009.48
|
| Dimethylallyl diphosphate | is_reported_donor_for | Abstract-reported NovQ precursor prenylation | DMAPP is the reported prenyl donor. | DOI:10.1038/ja.2009.48
|
| Abstract-reported NovQ precursor prenylation | forms_source_assigned_product | Source-assigned 3-dimethylallyl-4-hydroxyphenylpyruvate | The assigned product is an unglycosylated novobiocin precursor. This does not demonstrate mature owner synthesis or resolve its sugar stereochemistry. | DOI:10.1038/ja.2009.48
|
| Ozaki 2009 recombinant NovQ preparation | has_reported_alternative_substrate_scope | Abstract-reported alternative aromatic substrate scope | Reported C-C and C-O prenylations include phenylpropanoids, flavonoids and dihydroxynaphthalenes; examples include p-coumarate, caffeate and flavonoid B-ring substitution. Detailed yields and positional panels require the full primary results. | DOI:10.1038/ja.2009.48
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 15 nodes, 18 edges
NovQ fusion/preparation evidence from Ni 2019 (review #554), associated with the deposited NovQ precursor mechanism rather than new native proteins. The primary abstract, public figure previews and two supplementary gels were inspected; subscription main methods were not obtained. No complete construct, native strain or catalytic identity is inferred from soluble expression, SDS-PAGE or TEV processing.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Ni 2019 His6-NovQ preparation | was_tested_in | Ni 2019 NovQ fusion and co-expression study | The primary abstract identifies this partner in the NovQ expression study. Complete fusion sequence and preparation-specific strain provenance were not obtained; no native UniProt accession is assigned. | DOI:10.1007/s00449-018-2050-9
|
| Ni 2019 TrxA-NovQ fusion preparation | was_tested_in | Ni 2019 NovQ fusion and co-expression study | The primary abstract identifies this partner in the NovQ expression study. Complete fusion sequence and preparation-specific strain provenance were not obtained; no native UniProt accession is assigned. | DOI:10.1007/s00449-018-2050-9
|
| Ni 2019 GST-NovQ fusion preparation | was_tested_in | Ni 2019 NovQ fusion and co-expression study | The primary abstract identifies this partner in the NovQ expression study. Complete fusion sequence and preparation-specific strain provenance were not obtained; no native UniProt accession is assigned. | DOI:10.1007/s00449-018-2050-9
|
| Ni 2019 MBP-NovQ fusion preparation | was_tested_in | Ni 2019 NovQ fusion and co-expression study | The primary abstract identifies this partner in the NovQ expression study. Complete fusion sequence and preparation-specific strain provenance were not obtained; no native UniProt accession is assigned. | DOI:10.1007/s00449-018-2050-9
|
| E. coli BL21(DE3) expression host | was_used_in | Ni 2019 NovQ fusion and co-expression study | The study names this host. Host identity does not establish a native producer or the sequence of every fusion aliquot. | DOI:10.1007/s00449-018-2050-9
|
| E. coli Rosetta(DE3) expression host | was_used_in | Ni 2019 NovQ fusion and co-expression study | The study names this host. Host identity does not establish a native producer or the sequence of every fusion aliquot. | DOI:10.1007/s00449-018-2050-9
|
| Ni 2019 NovQ fusion and co-expression study | reports | Reported NovQ inclusion-body accumulation | The abstract reports predominantly insoluble NovQ in the pET28a system. This is condition-specific expression behavior, not loss of native catalytic function. | DOI:10.1007/s00449-018-2050-9
|
| Ni 2019 MBP-NovQ fusion preparation | has_reported_solubility_outcome | Abstract-reported MBP-associated solubility increase | The abstract describes soluble protein as approximately 70% of total cellular proteins. Retain that stated denominator; do not reinterpret it as 70% of NovQ, absolute yield, purity or catalytic conversion. The full quantitative methods were not obtained. | DOI:10.1007/s00449-018-2050-9
|
| Ni 2019 NovQ fusion and co-expression study | has_reported_expression_association | Reported rare-tRNA-associated expression change | The abstract associates greater rare-tRNA abundance with higher total and soluble recombinant protein. This does not demonstrate a particular translation bottleneck or supply a quantitative comparison for every construct. | DOI:10.1007/s00449-018-2050-9
|
| Ni 2019 NovQ fusion and co-expression study | has_reported_purification_outcome | Reported Ni-NTA purification outcome | The source reports greater than 90% recombinant-protein purity after Ni-NTA. Purity is distinct from soluble fraction, recovery and catalytic activity; it is not assigned to every fusion or lane. | DOI:10.1007/s00449-018-2050-9
|
| Ni 2019 MBP-NovQ fusion preparation | was_reportedly_processed_to | Ni 2019 TEV-processed MBP-NovQ material | In-vitro TEV cleavage is reported. Complete processed termini, residual tags, cleavage efficiency and activity recovery are not established by the inspected abstract. Processing is not assumed to regenerate native Q9L9F1. | DOI:10.1007/s00449-018-2050-9
|
| Ni 2019 NovQ fusion and co-expression study | includes_supplementary_comparison | Supplementary molecular-partner comparison | Figure S1 labels control, NovQ and molecular-partner groups with S/P lanes and NovQ arrows near 40 kDa. The caption does not identify the molecular partners or provide lane-normalization, replication or exact soluble percentages; the gel is not an enzyme-activity assay. | https://media.springernature.com/original/springer-static/esm/art%3A10.1007%2Fs00449-018-2050-9/MediaObjects/449_2018_2050_MOESM1_ESM.doc
|
| Ni 2019 NovQ fusion and co-expression study | includes_supplementary_comparison | Supplementary IPTG and temperature comparisons | Figure S2 names BL21(DE3). Its IPTG series is 0.1, 0.3, 0.5, 0.7 and 0.9 mM; temperature series is 20, 22, 24, 26 and 28 C. S/P lanes retain prominent arrowed bands near 40 kDa in P. These gel comparisons do not supply exact activity, a uniquely defined optimum or a complete factorial experiment. | https://media.springernature.com/original/springer-static/esm/art%3A10.1007%2Fs00449-018-2050-9/MediaObjects/449_2018_2050_MOESM1_ESM.doc
|
| Deposited native NovQ sequence reference | is_native_reference_for | Ni 2019 His6-NovQ preparation | UniProtKB:Q9L9F1 is the deposited native sequence reference for Ni 2019 His6-NovQ preparation (node 'his'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F1 |
| Deposited native NovQ sequence reference | is_native_reference_for | Ni 2019 TrxA-NovQ fusion preparation | UniProtKB:Q9L9F1 is the deposited native sequence reference for Ni 2019 TrxA-NovQ fusion preparation (node 'trx'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F1 |
| Deposited native NovQ sequence reference | is_native_reference_for | Ni 2019 GST-NovQ fusion preparation | UniProtKB:Q9L9F1 is the deposited native sequence reference for Ni 2019 GST-NovQ fusion preparation (node 'gst'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F1 |
| Deposited native NovQ sequence reference | is_native_reference_for | Ni 2019 MBP-NovQ fusion preparation | UniProtKB:Q9L9F1 is the deposited native sequence reference for Ni 2019 MBP-NovQ fusion preparation (node 'mbp'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F1 |
| Deposited native NovQ sequence reference | is_native_reference_for | Ni 2019 TEV-processed MBP-NovQ material | UniProtKB:Q9L9F1 is the deposited native sequence reference for Ni 2019 TEV-processed MBP-NovQ material (node 'cleaved'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F1 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 28 nodes, 37 edges
Nine NovQ E281 variants and bounded menadione-hydroquinol assays from Ni 2020 (review #554), linked to the NovQ precursor mechanism as engineered substrate-scope evidence. The full public ten-page supplement was inspected; main-article and correction access was limited to abstracts/metadata. Native sequence references, literal-primer models, actual preparations, docking and engineered-host product measurements are distinct. No native-product identity or full experimental sequence is inferred.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited native NovQ sequence reference | is_sequence_reference_for_primer_model | pMBPtev-NovQ parental DNA construct | The 323-aa native sequence supplies an explicit reconstruction reference, not proof of the full experimental construct. Table S2 names S. niveus for gene amplification without a strain number; the 16259 taxonomy conflict is not resolved or transferred to this sample by matching short primers. | https://www.uniprot.org/uniprotkb/Q9L9F1 |
| pMBPtev expression-vector design | is_reported_backbone_for | pMBPtev-NovQ parental DNA construct | Table S2 describes pMBPtev as pET30a-derived with MBP and a TEV site before the cloning site. Figure S1 shows the 6.4-kbp vector and NotI position. This schematic does not provide a complete plasmid sequence or every processed terminus. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
|
| pMBPtev-NovQ parental DNA construct | encodes_engineered_preparation | Ni 2020 pMBPtev-NovQ-derived WT assay preparation | Table S1 gives WT-F ATAAGAATGCGGCCGCAATGCCCGCACTCCCGATG and WT-R ATAAGAATGCGGCCGCTCAGTGGTGGTGGTGGTGGTGTCGGGCACCTCCGGTGAT (both 5-prime). Their native ends match the first and last 18 coding nt in both AF170880.4 and AB496950.1. The reverse-primer extension encodes six C-terminal histidines followed by stop: the reconstructed native-core-plus-His6 segment is 329 aa. This excludes unknown N-terminal vector/fusion sequence. The TEV site does not demonstrate tag cleavage in every kinetic aliquot, and cleavage would not by itself remove this C-terminal extension. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| pMBPtev-NovQ parental DNA construct | is_parental_reference_for | Ni 2020 NovQ point-mutant construct panel | The source names nine E281 substitutions in pMBPtev-derived constructs. All nine literal forward/reverse pairs are exact reverse complements and uniquely match native CDS nt826..858 with the designed mismatches. Models change only residue 281 in the native core; full plasmids and individual assayed aliquots were not independently sequenced. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| E. coli BL21(DE3) mutant expression host | was_expression_host_for | Ni 2020 NovQ point-mutant construct panel | Table S2 lists a BL21(DE3) strain for each pMBPtev mutant. The same table separately lists Rosetta(DE3)/pET28a-NovQ; do not assign that construct or host to all mutant preparations. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
|
| Ni 2020 pMBPtev-NovQ-derived WT assay preparation | was_compared_in | Supplementary menadione-hydroquinol rate series | Figure S5 shows a menadione-hydroquinol concentration series through 500 uM, with rate in nM/min, alongside the stated DMAPP cosubstrate. The supplement does not provide the complete assay recipe, protein loading, fixed DMAPP concentration or a fitted-parameter table. No exact Km/kcat or detection limit is digitized from the curve. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
|
| Menadione hydroquinol | was_varied_in | Supplementary menadione-hydroquinol rate series | The horizontal axis names the reduced aromatic substrate. It is not 4-HPP or an assay of the mature cathomycin structure. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
|
| Dimethylallyl diphosphate | was_reported_cosubstrate_in | Supplementary menadione-hydroquinol rate series | DMAPP is named in the captions. The displayed x-axis varies menadione hydroquinol, not DMAPP; a separate donor concentration series is not invented from those figures. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ-E281Q assay preparation | has_reported_activity_summary | Abstract-reported E281Q activity comparison | The primary abstract reports 2.05-fold activity and an aggregate 8.4-88.6% of WT range for the other variants. Without the full kinetic table, this is not assigned as a specific kcat/Km ratio, separate substrate-dependent constants or a value for any particular other mutant. | DOI:10.1007/s00253-020-10470-w
|
| Ni 2020 NovQ point-mutant construct panel | tests_functional_consequences_near_hypothesis | Docking-based NovQ substrate-contact hypothesis | The study motivates E281 substitutions using substrate docking; Figure S2 is a docking-energy distribution. This is computational contact evidence, not a solved NovQ-substrate complex or direct proton-transfer observation. The publisher records a Figure 1 correction dated 2020-04-14; its full correction and main article were not obtained. No uninspected atom-level contact map or corrected Figure 1 geometry is asserted. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Supplementary menadione-hydroquinol rate series | has_study_product_assignment | Source-assigned C3-prenylated menadione product | The study assigns C3-prenylated menadione. This is a surrogate-product claim; it does not establish synthesis of mature novobiocin/cathomycin or the redox state/order of every catalytic intermediate. | DOI:10.1007/s00253-020-10470-w
|
| Supplementary product MS, UV and proton-NMR evidence | supports_source_product_assignment | Source-assigned C3-prenylated menadione product | Figure S3 shows an MS peak labelled m/z 241.1222 and UV markers at 244, 248, 261 and 269 nm. Figure S4 supplies a proton-NMR spectrum in CDCl3 with TMS. These are the source product-characterization figures, not separate structural determinations for each mutant or fermentation sample. No independent full spectral assignment or exact chemical record is constructed here. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
|
| pMVA engineered precursor-supply construct | was_present_in | Ni 2020 S1 host: pMVA only | Table S2 identifies S1 as BL21(DE3) with pMVA alone. pMVA lists atoB-HMGS-HMGR-ERG12-ERG8-MVD1-idi on pCDFDuet-1. The gene list does not establish exact protein accessions, expression stoichiometry or a measured intracellular donor concentration. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| pMVA engineered precursor-supply construct | was_present_in | Ni 2020 S2 host: pMVA plus pMBPtevQ | Table S2 identifies S2 as BL21(DE3) carrying pMVA plus pMBPtevQ. This is an engineered precursor-supply context, not the native BGC. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ-E281Q assay preparation | was_construct_encoded_in | Ni 2020 S2 host: pMVA plus pMBPtevQ | pMBPtevQ is the E281Q expression construct in S2. This associates the engineered sequence design with the host; it does not measure protein abundance or show in-vivo TEV processing. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ-E281Q assay preparation | was_construct_encoded_in | Ni 2020 S3 host: pMBPtevQ only | S3 carries pMBPtevQ without pMVA. Its definition is distinct from the S2 coexpression strain; the supplement alone does not supply a quantitative product titer for S3. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| pMBPtev expression-vector design | was_present_in | Ni 2020 S4 host: pMBPtev vector | S4 carries the pMBPtev vector. This records the source comparator design, not a measured zero-product result. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 S2 host: pMVA plus pMBPtevQ | matches_reported_production_configuration | Reported engineered-host prenylated-menadione output | The abstract reports up to 4.7 mg/L C3-prenylated menadione for E281Q expression plus engineered MVA supply and exogenous aromatic substrate. S2 matches that listed configuration. This is an author-reported engineered-host titer, not enzyme specific activity, native antibiotic flux or a de-novo-from-glucose yield; detailed culture and uncertainty were not obtained. | DOI:10.1007/s00253-020-10470-w |
| Reported engineered-host prenylated-menadione output | concerns_source_assigned_product | Source-assigned C3-prenylated menadione product | The fermentation claim concerns a fed-substrate prenylation product, not the complete cathomycin molecule or its unresolved sugar stereocentre. | DOI:10.1007/s00253-020-10470-w
|
| Ni 2020 NovQ point-mutant construct panel | generated_expression_construct_for | Ni 2020 NovQ-E281Q assay preparation | Table S2 names BL21-MBP-NovQ-E281Q with pMBPtevQ. Forward primer 5-prime GACTACGTCAAGGCCCAACGCAGCTACACCGGG; reverse primer 5-prime CCCGGTGTAGCTGCGTTGGGCCTTGACGTAGTC. The deposited-template model changes only native E281 to Q. Tag, junction and cleavage state are not fully specified for the assayed protein; no native accession is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Ni 2020 NovQ-E281Q assay preparation | has_displayed_rate_series_in | Supplementary menadione-hydroquinol rate series | Figure S6 panel a is labelled NovQ E281Q and plots rate against menadione hydroquinol through 500 uM. Panels have different vertical scales. The caption does not define error bars or replicate count; no exact digitized rate, mutant kinetic constant, binding affinity or universal activity ranking is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ point-mutant construct panel | generated_expression_construct_for | Ni 2020 NovQ-E281D assay preparation | Table S2 names BL21-MBP-NovQ-E281D with pMBPtevD. Forward primer 5-prime GACTACGTCAAGGCCGACCGCAGCTACACCGGG; reverse primer 5-prime CCCGGTGTAGCTGCGGTCGGCCTTGACGTAGTC. The deposited-template model changes only native E281 to D. Tag, junction and cleavage state are not fully specified for the assayed protein; no native accession is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Ni 2020 NovQ-E281D assay preparation | has_displayed_rate_series_in | Supplementary menadione-hydroquinol rate series | Figure S6 panel b is labelled NovQ E281D and plots rate against menadione hydroquinol through 500 uM. Panels have different vertical scales. The caption does not define error bars or replicate count; no exact digitized rate, mutant kinetic constant, binding affinity or universal activity ranking is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
|
| Ni 2020 NovQ point-mutant construct panel | generated_expression_construct_for | Ni 2020 NovQ-E281T assay preparation | Table S2 names BL21-MBP-NovQ-E281T with pMBPtevT. Forward primer 5-prime GACTACGTCAAGGCCACACGCAGCTACACCGGG; reverse primer 5-prime CCCGGTGTAGCTGCGTGTGGCCTTGACGTAGTC. The deposited-template model changes only native E281 to T. Tag, junction and cleavage state are not fully specified for the assayed protein; no native accession is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Ni 2020 NovQ-E281T assay preparation | has_displayed_rate_series_in | Supplementary menadione-hydroquinol rate series | Figure S6 panel c is labelled NovQ E281T and plots rate against menadione hydroquinol through 500 uM. Panels have different vertical scales. The caption does not define error bars or replicate count; no exact digitized rate, mutant kinetic constant, binding affinity or universal activity ranking is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ point-mutant construct panel | generated_expression_construct_for | Ni 2020 NovQ-E281S assay preparation | Table S2 names BL21-MBP-NovQ-E281S with pMBPtevS. Forward primer 5-prime GACTACGTCAAGGCCAGCCGCAGCTACACCGGG; reverse primer 5-prime CCCGGTGTAGCTGCGGCTGGCCTTGACGTAGTC. The deposited-template model changes only native E281 to S. Tag, junction and cleavage state are not fully specified for the assayed protein; no native accession is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Ni 2020 NovQ-E281S assay preparation | has_displayed_rate_series_in | Supplementary menadione-hydroquinol rate series | Figure S6 panel d is labelled NovQ E281S and plots rate against menadione hydroquinol through 500 uM. Panels have different vertical scales. The caption does not define error bars or replicate count; no exact digitized rate, mutant kinetic constant, binding affinity or universal activity ranking is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ point-mutant construct panel | generated_expression_construct_for | Ni 2020 NovQ-E281A assay preparation | Table S2 names BL21-MBP-NovQ-E281A with pMBPtevA. Forward primer 5-prime GACTACGTCAAGGCCGCACGCAGCTACACCGGG; reverse primer 5-prime CCCGGTGTAGCTGCGTGCGGCCTTGACGTAGTC. The deposited-template model changes only native E281 to A. Tag, junction and cleavage state are not fully specified for the assayed protein; no native accession is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Ni 2020 NovQ-E281A assay preparation | has_displayed_rate_series_in | Supplementary menadione-hydroquinol rate series | Figure S6 panel e is labelled NovQ E281A and plots rate against menadione hydroquinol through 500 uM. Panels have different vertical scales. The caption does not define error bars or replicate count; no exact digitized rate, mutant kinetic constant, binding affinity or universal activity ranking is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ point-mutant construct panel | generated_expression_construct_for | Ni 2020 NovQ-E281V assay preparation | Table S2 names BL21-MBP-NovQ-E281V with pMBPtevV. Forward primer 5-prime GACTACGTCAAGGCCGTACGCAGCTACACCGGG; reverse primer 5-prime CCCGGTGTAGCTGCGTACGGCCTTGACGTAGTC. The deposited-template model changes only native E281 to V. Tag, junction and cleavage state are not fully specified for the assayed protein; no native accession is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Ni 2020 NovQ-E281V assay preparation | has_displayed_rate_series_in | Supplementary menadione-hydroquinol rate series | Figure S6 panel f is labelled NovQ E281V and plots rate against menadione hydroquinol through 500 uM. Panels have different vertical scales. The caption does not define error bars or replicate count; no exact digitized rate, mutant kinetic constant, binding affinity or universal activity ranking is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ point-mutant construct panel | generated_expression_construct_for | Ni 2020 NovQ-E281M assay preparation | Table S2 names BL21-MBP-NovQ-E281M with pMBPtevM. Forward primer 5-prime GACTACGTCAAGGCCATGCGCAGCTACACCGGG; reverse primer 5-prime CCCGGTGTAGCTGCGCATGGCCTTGACGTAGTC. The deposited-template model changes only native E281 to M. Tag, junction and cleavage state are not fully specified for the assayed protein; no native accession is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Ni 2020 NovQ-E281M assay preparation | has_displayed_rate_series_in | Supplementary menadione-hydroquinol rate series | Figure S6 panel g is labelled NovQ E281M and plots rate against menadione hydroquinol through 500 uM. Panels have different vertical scales. The caption does not define error bars or replicate count; no exact digitized rate, mutant kinetic constant, binding affinity or universal activity ranking is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ point-mutant construct panel | generated_expression_construct_for | Ni 2020 NovQ-E281R assay preparation | Table S2 names BL21-MBP-NovQ-E281R with pMBPtevR. Forward primer 5-prime GACTACGTCAAGGCCCGACGCAGCTACACCGGG; reverse primer 5-prime CCCGGTGTAGCTGCGTCGGGCCTTGACGTAGTC. The deposited-template model changes only native E281 to R. Tag, junction and cleavage state are not fully specified for the assayed protein; no native accession is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Ni 2020 NovQ-E281R assay preparation | has_displayed_rate_series_in | Supplementary menadione-hydroquinol rate series | Figure S6 panel h is labelled NovQ E281R and plots rate against menadione hydroquinol through 500 uM. Panels have different vertical scales. The caption does not define error bars or replicate count; no exact digitized rate, mutant kinetic constant, binding affinity or universal activity ranking is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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| Ni 2020 NovQ point-mutant construct panel | generated_expression_construct_for | Ni 2020 NovQ-E281K assay preparation | Table S2 names BL21-MBP-NovQ-E281K with pMBPtevK. Forward primer 5-prime GACTACGTCAAGGCCAAACGCAGCTACACCGGG; reverse primer 5-prime CCCGGTGTAGCTGCGTTTGGCCTTGACGTAGTC. The deposited-template model changes only native E281 to K. Tag, junction and cleavage state are not fully specified for the assayed protein; no native accession is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf |
| Ni 2020 NovQ-E281K assay preparation | has_displayed_rate_series_in | Supplementary menadione-hydroquinol rate series | Figure S6 panel i is labelled NovQ E281K and plots rate against menadione hydroquinol through 500 uM. Panels have different vertical scales. The caption does not define error bars or replicate count; no exact digitized rate, mutant kinetic constant, binding affinity or universal activity ranking is assigned. | https://static-content.springer.com/esm/art%3A10.1007%2Fs00253-020-10470-w/MediaObjects/253_2020_10470_MOESM1_ESM.pdf
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Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 18 nodes, 18 edges
NovO native sequence, historical taxonomy, abstract-limited 2005 precursor chemistry and separate Sadler 2017 preparations. Native Q9L9F3 is a deposited sequence reference, not an engineered assay protein. Review #555. PDF locators refer to the 60-page accepted manuscript/SI, not final journal pagination; SI printed pages are seven lower. The cathomycin owner retains its unspecified sugar stereocentre.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novO locus from NCIMB 9219 | The owner links BGC0000834.5 and AF170880.4. novO is 17538..18230(+) with a 693-nt CDS including stop. This is a locus association, not a new equivalence to fully specified novobiocin. | https://mibig.secondarymetabolites.org/go/BGC0000834.5 |
| Deposited novO locus from NCIMB 9219 | encodes_native_protein | Deposited native NovO sequence reference | Table-11 CDS-aware translation matches AAF67508.2 and all 230 residues of Q9L9F3. Current GenBank source is S. niveus NCIMB 9219, taxon 193462. The native accession is not assigned to tagged or mutant samples. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited native NovO sequence reference | has_identical_deposited_sequence_to | NovO deposit from strain 40292 | JN606326.1/AEP25522.1 has a 1..693(+) CDS whose translated 230-aa protein is identical. Protein-sequence equality does not establish strain synonymy or the provenance of any assayed aliquot. | https://www.ncbi.nlm.nih.gov/nuccore/JN606326.1 |
| NovO deposit from strain 40292 | has_unresolved_source_taxonomy | NovO source-taxon and historical strain disagreement | JN606326.1 names Actinoalloteichus caeruleus taxon 2893586, strain 40292, while its definition retains Streptomyces caeruleus. UniProt uses S. niveus and historical DSM 40292/NCIMB 11891 reference metadata. These labels are not silently reconciled with AF170880.4 NCIMB 9219 and do not change the owner producer. | https://www.ncbi.nlm.nih.gov/nuccore/JN606326.1 |
| Deposited native NovO sequence reference | is_database_reference_for_studied_enzyme | Pacholec 2005 recombinant NovO preparation | UniProt links NovO function to Pacholec 2005. Its primary abstract reports E. coli expression and purification, but full construct sequence and methods were not obtained; no native accession is assigned as the exact recombinant preparation identity. | https://www.uniprot.org/uniprotkb/Q9L9F3 |
| Pacholec 2005 recombinant NovO preparation | catalyzes_reported_methylation | Abstract-reported NovO precursor C-methylation | The abstract reports SAM-dependent aminocoumarin C-methylation after ring construction and amide ligation. No numerical kinetic constant, loading or tag state is inferred from the 2017 study. | DOI:10.1021/bi051599o
|
| Unmethylated ligated aminocoumarin precursor | is_reported_acceptor_for | Abstract-reported NovO precursor C-methylation | UniProt calls the substrate 8-desmethylnovobiocic acid (RHEA:36651); the 2005 abstract describes the NovL-ligated product as novobiocic acid. Both concern an unglycosylated precursor, not the 2017 benzamide surrogate or mature owner structure. | DOI:10.1021/bi051599o |
| Abstract-reported NovO precursor C-methylation | forms_reported_precursor | C8-methylated novobiocic-acid precursor | The source places a C-methyl adjacent to the phenolic oxygen. This does not demonstrate mature cathomycin synthesis or resolve the owner sugar stereocentre. | DOI:10.1021/bi051599o
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| Pacholec 2005 recombinant NovO preparation | has_reported_negative_substrate_results | Reported nonacceptance of earlier pathway intermediates | The abstract reports nonacceptance of NovH-tethered tyrosyl-derived S-pantetheinyl intermediates and subsequently released free aminocoumarin. Assay-specific detection limits were not supplied. | DOI:10.1021/bi051599o
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| Abstract-reported NovO precursor C-methylation | precedes_reported_glycosylation | Source-supported methylation before glycosylation | The reported NovO step precedes NovM O-glycosylation. This is the source pathway interpretation, not native-cell flux measurement or evidence that every accepted analogue follows this order. | DOI:10.1021/bi051599o
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| Deposited native NovO sequence reference | is_sequence_reference_for | Sadler codon-optimized pET26b(+)-NovO-His6 DNA construct | The 2017 gene was synthesized by GenScript with codon optimization and a C-terminal His6, then cloned into pET26b(+) via NdeI/XhoI. The native DNA is not its actual template. The PDB construct core matches the native protein, but the complete synthetic DNA and individual mutant plasmid sequences were not independently obtained. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf |
| Sadler codon-optimized pET26b(+)-NovO-His6 DNA construct | encodes_engineered_protein | Sadler C-terminal His6 NovO WT preparation | The reported C-terminal His6 makes this distinct from native NovO. 5MGZ deposits a 230-aa native core plus six histidines (236 aa); that structural deposit does not sequence every assay preparation. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf |
| E. coli BL21(DE3) NovO expression host | expressed | Sadler C-terminal His6 NovO WT preparation | The standard protocol uses BL21(DE3), Magic Media autoinduction, 50 ug/mL kanamycin, growth at 30 C to OD about 2, then 18 C overnight. TOP10 was used for cloning, not identified as the production host. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler codon-optimized pET26b(+)-NovO-His6 DNA construct | was_used_for_labelled_preparation | Sadler SeMet-labelled NovO-His6 preparation | BL21(DE3) expression for SeMet labelling used a distinct minimal-medium protocol, 50 ug/mL selenomethionine and 100 uM IPTG at 18 C. This is a chemically labelled tagged preparation, not a new native allele. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler C-terminal His6 NovO WT preparation | has_reported_purification_readout | Sadler NovO purification gels | HisTrap affinity purification is described for WT and mutants. The mutant gel on PDF p.36 labels F14M/F14L/R24F/R116K/R116L/N117A/H120N/R121L/R121Q/Y216F/WT. H120A is absent; R121Q is an abbreviated lane label, not evidence against the activity table double-mutant label. Bands do not establish complete sequence, exact purity or equal active fraction. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler SeMet-labelled NovO-His6 preparation | has_reported_purification_readout | Sadler NovO purification gels | SeMet material received additional Superdex 75 SEC after HisTrap; the lower gel labels affinity and SEC pools/concentrates. This purification SEC is not a calibrated solution-stoichiometry assay. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler codon-optimized pET26b(+)-NovO-His6 DNA construct | has_listed_primer_design | R24M primer design without an endpoint row | Listed forward 5-prime GGCTCGCAAGCATCGCACATGTATGACGAATTTGTT; reverse 5-prime AACAAATTCGTCATACATGTGCGATGCTTGCGAGCC. The pair is reverse-complementary and its local translated peptide matches the named substitution. This name occurs only in the primer list in the inspected manuscript/SI text and is not an endpoint-table or displayed purification-gel entry; no activity or purified-protein outcome is invented. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf |
| Sadler codon-optimized pET26b(+)-NovO-His6 DNA construct | has_listed_primer_design | H120F primer design without an endpoint row | Listed forward 5-prime GTGAGTCGTAATGCCTTTTTCCGTCTGACCCGCCTG; reverse 5-prime CAGGCGGGTCAGACGGAAAAAGGCATTACGACTCAC. The pair is reverse-complementary and its local translated peptide matches the named substitution. This name occurs only in the primer list in the inspected manuscript/SI text and is not an endpoint-table or displayed purification-gel entry; no activity or purified-protein outcome is invented. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 22 nodes, 33 edges
Eleven NovO engineered variants in the Sadler 2017 24-hour benzamide-surrogate assay, with WT and no-MT controls. Lysate, double-mutant identity, censored values, loading conflict and preparative recovery remain explicit. Review #555. PDF locators refer to the 60-page accepted manuscript/SI, not final journal pagination; SI printed pages are seven lower. The cathomycin owner retains its unspecified sugar stereocentre.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Sadler codon-optimized NovO-His6 parent construct | encodes_engineered_preparation | Sadler NovO-His6 WT assay preparation | The source uses a codon-optimized pET26b(+) gene with C-terminal His6. WT means this recombinant assay comparator, not untagged native NovO. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-His6 WT assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports WT 98% conversion after 24 h. This is HPLC area/area% at 320 nm, not 98% relative to a 100%-normalized WT, an initial turnover rate or independently calibrated molar conversion. No replicate count or uncertainty accompanies the endpoint table. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)benzamide | is_acceptor_for | Twenty-four-hour surrogate methylation assay | Substrate 6 is the synthetic benzamide coumarin, not native 8-desmethylnovobiocic acid or mature cathomycin. The assay lists 100 uM acceptor, 2 mM SAM, 0.1 mg/mL BSA, 50 mM sodium phosphate pH 6.5, total 0.5 mL, 35 C and 1000 rpm for 24 h, followed by 80 C for 15 min to quench. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| S-Adenosyl-L-methionine donor | is_donor_for | Twenty-four-hour surrogate methylation assay | SAM is directly supplied at a stated 2 mM in this endpoint assay. Do not transfer SalL-mediated donor-supply methods from the separate KIE assay. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Twenty-four-hour surrogate methylation assay | forms_source_assigned_product | C8-methylated benzamide surrogate product | The HPLC product is assigned as N-(4,7-dihydroxy-8-methyl-2-oxo-2H-chromen-3-yl)benzamide (4a). Preparative characterization supports the source assignment but is not a separate structural determination of every mutant endpoint. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| No-methyltransferase negative control | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | The no-MT control is reported as 0% area conversion. This is the source endpoint, not a measured absolute zero or a stated detection limit. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Twenty-four-hour surrogate methylation assay | has_unresolved_loading_description | Unresolved enzyme-loading units | Section 12 reports 250 ug purified enzyme in 0.5 mL and a final 1.9 uM concentration. With the source 26.23-kDa theoretical mass (PDF p.36), the mass/volume implies about 19.06 uM, roughly tenfold higher. Neither value is silently selected as actual loading; no new enzyme-normalized rate is calculated. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf |
| Sadler codon-optimized NovO-His6 parent construct | was_expressed_for_lysate_preparation | Preparative NovO-expressing cell lysate | The preparative experiment uses E. coli lysate from NovO overexpression, distinct from purified WT in the endpoint comparison. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Preparative NovO-expressing cell lysate | was_used_in | Preparative surrogate methylation and isolated recovery | The procedure starts with 40 mL lysate, 20 mg substrate and 72 mg SAM, then adds another 72 mg SAM after 24 h and incubates for a further 16 h. It reports 90% HPLC conversion and 14 mg isolated product (67% yield). Conversion and isolated recovery are different readouts, not native antibiotic titers or a WT-versus-mutant comparison. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Preparative surrogate methylation and isolated recovery | yielded_characterized_product | C8-methylated benzamide surrogate product | The source provides proton/carbon NMR and HRMS (calculated [M+H]+ 312.0868, found 312.0866) for the isolated surrogate. No new exact-structure record or independent full spectral assignment is made. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-F14M assay preparation | Literal forward 5-prime GGTTCCGAAGCAGAAGCAATGCACCGCATGGGCTCG; reverse 5-prime CGAGCCCATGCGGTGCATTGCTTCTGCTTCGGAACC. The reverse-complement pair encodes the named local peptide change. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler NovO-F14M assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports 19% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-F14L assay preparation | Literal forward 5-prime GGTTCCGAAGCAGAAGCACTCCACCGCATGGGCTCG; reverse 5-prime CGAGCCCATGCGGTGGAGTGCTTCTGCTTCGGAACC. The reverse-complement pair encodes the named local peptide change. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler NovO-F14L assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports 30% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-R24F assay preparation | Literal forward 5-prime GGCTCGCAAGCATCGCACUTCTATGACGAATTTGTT; reverse 5-prime AACAAATTCGTCATAGAAGTGCGATGCTTGCGAGCC. The forward primer contains U as printed; it is not silently rewritten as DNA T. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler NovO-R24F assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports 15% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-R116L assay preparation | The activity table and gel name this mutant, but the inspected SDM primer list does not give its pair. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler NovO-R116L assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports 1% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. Retain this numeric/censored value despite main prose describing complete loss for this position; <1% is not a measured zero. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-R116K assay preparation | The activity table and gel name this mutant, but the inspected SDM primer list does not give its pair. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler NovO-R116K assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports <1% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. Retain this numeric/censored value despite main prose describing complete loss for this position; <1% is not a measured zero. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-N117A assay preparation | Literal forward 5-prime CTGGTCGTGAGTCGTGCTGCCTTTCACCGTCTGACC; reverse 5-prime GGTCAGACGGTGAAAGGCAGCACGACTCACGACCAG. The reverse-complement pair encodes the named local peptide change. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler NovO-N117A assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports <1% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. Retain this numeric/censored value despite main prose describing complete loss for this position; <1% is not a measured zero. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-H120A assayed in cell lysate | Literal forward 5-prime GTGAGTCGTAATGCCTTTGCCCGTCTGACCCGCCTG; reverse 5-prime CAGGCGGGTCAGACGGGCAAAGGCATTACGACTCAC. The reverse-complement pair encodes the named local peptide change. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-H120A assayed in cell lysate | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports 0% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. Cell lysate was used because of poor soluble expression, so the 0% result does not isolate catalytic impairment from protein abundance/folding. The purified-enzyme loading cannot be assumed for this lysate. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf |
| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-H120N assay preparation | Literal forward 5-prime GTGAGTCGTAATGCCTTTAACCGTCTGACCCGCCTG; reverse 5-prime CAGGCGGGTCAGACGGTTAAAGGCATTACGACTCAC. The reverse-complement pair encodes the named local peptide change. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-H120N assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports <1% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. Retain this numeric/censored value despite main prose describing complete loss for this position; <1% is not a measured zero. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-R121L assay preparation | Literal forward 5-prime CGTAATGCCTTTCACCTTCTGACCCGCCTGCCG; reverse 5-prime CGGCAGGCGGGTCAGAAGGTGAAAGGCATTACG. The reverse-complement pair encodes the named local peptide change. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-R121L assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports <1% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. Retain this numeric/censored value despite main prose describing complete loss for this position; <1% is not a measured zero. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-R121Q/G108D assay preparation | Literal forward 5-prime CGTAATGCCTTTCACCAGCTGACCCGCCTGCCG; reverse 5-prime CGGCAGGCGGGTCAGCTGGTGAAAGGCATTACG. The reverse-complement pair encodes the named local peptide change. The measured row is R121Q/G108D, not a clean R121Q single mutant. The R121Q primer does not encode the remote G108D change; the gel abbreviates the lane R121Q. The actual double mutant is retained as reported. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-R121Q/G108D assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports <1% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. Retain this numeric/censored value despite main prose describing complete loss for this position; <1% is not a measured zero. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler codon-optimized NovO-His6 parent construct | is_reported_parent_for | Sadler NovO-Y216F assay preparation | Literal forward 5-prime GTGTGGATCGATGACCAAGGCTTCGGTGTGCCGACGGTT; reverse 5-prime AACCGTCGGCACACCGAAGCCTTGGTCATCGATCCACAC. The reverse-complement pair encodes the named local peptide change. The authors report sequencing mutant genes. Full experimental plasmids were not independently obtained; short peptide models do not establish complete sequence identity. No native accession is assigned. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-Y216F assay preparation | has_measured_endpoint_in | Twenty-four-hour surrogate methylation assay | Table 2 reports 20% HPLC area conversion at the stated 24-hour endpoint. No replicate count or uncertainty is supplied; this is not kcat, binding affinity or native precursor turnover. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Deposited native NovO sequence reference | is_native_reference_for | Sadler NovO-His6 WT assay preparation | UniProtKB:Q9L9F3 is the deposited native sequence reference for Sadler NovO-His6 WT assay preparation (node 'wt'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F3 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 10 edges
Observed NovO-SAH structure and deposited homodimer are kept distinct from coumarin/SAM docking, substrate-entry and catalytic hypotheses in Sadler 2017. Review #555. PDF locators refer to the 60-page accepted manuscript/SI, not final journal pagination; SI printed pages are seven lower. The cathomycin owner retains its unspecified sugar stereocentre.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited native NovO sequence reference | is_core_sequence_reference_for | Sadler SeMet-labelled NovO-His6 crystallization preparation | The deposited construct is exactly Q9L9F3 residues 1-230 plus C-terminal HHHHHH. The source describes SeMet labelling for phasing; native Q9L9F3 is not the full labelled/tagged preparation. | https://www.uniprot.org/uniprotkb/Q9L9F3 |
| Sadler SeMet-labelled NovO-His6 crystallization preparation | was_used_for_structure_determination | Deposited 5MGZ NovO-SAH structural model | The source reports single-wavelength anomalous dispersion with 12 SeMet sites, two protein molecules in the asymmetric unit and 1.9-A resolution. The current 5MGZ coordinate model uses ordinary MET/S atoms and contains no Se atoms. Coordinate atom naming does not negate source-described SeMet phasing or measure labelling occupancy. Both chains model residues 1-230; all six tag residues per chain are unmodeled, not shown to have been cleaved. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf |
| Deposited 5MGZ NovO-SAH structural model | has_assigned_biological_assembly | Author/PISA-assigned NovO homodimer | 5MGZ assembly 1 is dimeric, assigned by authors and PISA, with identity-operation chains A/B. This supports the deposited assembly model, not direct native-cell stoichiometry or a newly measured solution equilibrium. The manuscript cites an earlier solution-dimer study; that separate full study was not inspected here. | https://www.rcsb.org/structure/5MGZ |
| Deposited 5MGZ NovO-SAH structural model | contains_observed_ligand | S-Adenosyl-L-homocysteine in 5MGZ | Each chain contains one 26-heavy-atom SAH. The deposit also contains glycerol, acetate and water, not a bound coumarin or SAM. SI omit maps describe defined SAH conformations in both chains. The source used 12 mg/mL protein preincubated with 2 mM SAH; soaking/solution concentrations are not binding constants. | https://files.rcsb.org/download/5MGZ.cif |
| S-Adenosyl-L-homocysteine in 5MGZ | has_observed_polar_contacts | Observed NovO-SAH polar contact geometry | Independent coordinate distances in chains A/B are E4-OE1 to SAH-N6 2.978/2.994 A, D96-OD1 to N6 2.832/2.812 A, D70-OD1 to O2-prime 2.688/2.639 A, and R116-NH1 to O 2.782/2.810 A. These support the polar geometry described by Figure 1 and SI Figure S4, not observed proton transfer, SAM chemistry or a coumarin contact. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf |
| Deposited 5MGZ NovO-SAH structural model | shows_local_alternate_conformations | Chain-A R24 alternate conformations | The source emphasizes flexible R24 and illustrates chain A. The deposit has A/B alternate atom locations for chain-A R24 only; chain B has no alternate R24 locations. Alternate locations and B-factors are not direct measurements of a substrate-entry trajectory. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf |
| Chain-A R24 alternate conformations | motivates_proposed_gatekeeping | Proposed R24 substrate-entry role | A substrate-entry role is inferred from location, flexibility and mutagenesis, not directly observed transport through a gate. R24F endpoint impairment does not uniquely establish the route. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Deposited 5MGZ NovO-SAH structural model | served_as_template_for | MOE-derived coumarin and SAM binding hypothesis | Attempts to obtain a coumarin-bound crystal failed. MOE retained SAH, used Protonate3D at pH 7 and selected a docking model partly by coumarin C8 proximity to SAH sulfur. Adding a modeled methyl gave a reported 2.59-A C8 distance and 109-degree orientation. These are modeled values, not contacts measured in 5MGZ. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
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| Synthetic coumarin benzamide docking substrate | was_modelled_in | MOE-derived coumarin and SAM binding hypothesis | The main text and SI model methods name substrate 6, while the SI Figure S7 caption calls it 4a. Preserve that caption-label difference; neither establishes an observed enzyme-substrate or enzyme-product complex. No exact ligand identifier is assigned from the docking picture. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| MOE-derived coumarin and SAM binding hypothesis | supports_proposed_catalytic_model | Proposed H120/R121-assisted C8 methylation mechanism | The authors propose H120 deprotonation of the substrate 7-OH, R121-assisted stabilization/positioning and C8 methyl transfer from SAM. R116/N117, F14 and Y216 roles also derive from docking and mutational effects. These data do not directly observe a proton trajectory or uniquely separate binding, folding and chemistry; the source also allows R116/N117 to stabilize the phenoxide. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 13 nodes, 14 edges
Two additional NovO lysine variants, the WT comparator and auxiliary SalL in Sadler 2017 pH, substrate-analogue and coupled isotope experiments. Reported measurements remain separate from uncertain scale, substrate-axis identity and mechanistic inference. Review #555. PDF locators refer to the 60-page accepted manuscript/SI, not final journal pagination; SI printed pages are seven lower. The cathomycin owner retains its unspecified sugar stereocentre.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Sadler NovO-His6 WT kinetic comparator | was_comparator_in | NovO WT and lysine-variant pH comparison | Figure 3b compares WT, H120K and R121K over pH 5-8 using benzamide substrate 6. The y-axis is percent conversion, not initial turnover or binding affinity. No per-point numerical table, uncertainty or replicate count is supplied; exact points are not digitized. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-H120K assay preparation | has_displayed_pH_response_in | NovO WT and lysine-variant pH comparison | H120K is a separately named lysine mutant, not H120A or H120N. The source text reports inactivity over pH 5-7; the displayed curve remains near baseline through pH 8. This is an assay-bound result, not a proven functional-null enzyme. The inspected SDM list does not provide H120K primers or an individual preparation gel. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-R121K assay preparation | has_displayed_pH_response_in | NovO WT and lysine-variant pH comparison | The source describes R121K as retaining approximately 20% of WT activity above pH 6.5. This approximation is not a precise kinetic constant or a fitted catalytic pKa. The inspected SDM list does not provide R121K primers or an individual preparation gel. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-His6 WT kinetic comparator | was_tested_against | Four 7-position substrate-analogue comparisons | Replacing the 7-OH of substrate 6 with Cl, NO2, NH2 or OMe (S1-S4) yielded 0% reported conversion in SI Scheme S1 under the 24-h endpoint conditions, versus 98% for substrate 6. These are condition-specific HPLC negatives with no stated detection limit, not universal exclusion of all related substrates. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Sadler NovO-His6 WT kinetic comparator | was_used_in | SalL-assisted isotope donor-supply assay | KIE reactions were run in triplicate at 200 uL: the general recipe lists 0.5 mM substrate 6, 2 mM ClDA, 2.5 mM methionine or isotopic methionine and 0.1 mg/mL SalL in 50 mM phosphate pH 6.5, incubated 2 h at 37 C and 750 rpm. Addition of NovO to 0.5 mg/mL began the methylation phase; aliquots were quenched after 3 min at 97 C for 10 min, then analyzed by HPLC at 320 nm. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| SalL preparation used for coupled donor supply | was_used_for_donor_supply_in | SalL-assisted isotope donor-supply assay | SalL is the named enzyme in the ClDA/methionine preincubation. It is an auxiliary assay protein, not assigned as a native novobiocin BGC component. This source does not supply a full SalL construct sequence; no convenient native accession is substituted for it. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Me-SAM, carbon-13 SAM and deuterated SAM assay conditions | define_isotopic_conditions_for | SalL-assisted isotope donor-supply assay | Methionine, methionine-13C and methionine-d3 supply the three conditions labelled Me-SAM, 13CH3-SAM and CD3-SAM. The supplied precursor concentration is not a direct measurement of generated SAM concentration, purity or stereoisomer composition. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| SalL-assisted isotope donor-supply assay | yielded_reported_kinetic_fits | Reported apparent kinetic fits with unresolved scale conflict | Table 1 gives Km values 43.7 +/- 2.8, 41.6 +/- 5.1 and 52.8 +/- 5.3 uM for Me-SAM/13CH3-SAM/CD3-SAM-labelled conditions, with kcat 2.1e-3 +/- 0.0004, 1.7e-3 +/- 0.0008 and 2.0e-3 +/- 0.0009 s^-1. Nearby prose instead states 20.1e-3, 17.0e-3 and 20.1e-3 s^-1, approximately tenfold higher. Both are preserved as an accepted-manuscript conflict. SI Figure S10 plots initial velocity in nmol/min versus generic substrate concentration in mM; the fixed general recipe does not explain which species was varied. No unique acceptor/donor Km, binding affinity, corrected turnover or uncertainty type is inferred. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Reported apparent kinetic fits with unresolved scale conflict | has_reported_primary_isotope_comparison | Reported primary carbon-13 kinetic isotope effect | The source reports a primary KIE of 1.22 +/- 0.04. It is retained as reported, not recomputed from rounded and inconsistent turnover entries or promoted to a directly observed transition-state structure. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Reported apparent kinetic fits with unresolved scale conflict | has_reported_secondary_isotope_comparison | Reported nonsignificant secondary isotope effect | The reported secondary KIE is 1.03 +/- 0.03 and is explicitly described as not statistically significant. No significance or new confidence interval is manufactured from the rounded table. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Reported primary carbon-13 kinetic isotope effect | supports_proposed_rate_limitation | Proposed substrate activation and methyl-transfer limitation | The authors interpret the primary effect as consistent with rate-limiting methyl transfer and an SN2-like transition state. This is a mechanistic inference from a coupled assay, not direct observation or a uniquely proven microscopic rate-limiting step. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| NovO WT and lysine-variant pH comparison | supports_proposed_substrate_activation | Proposed substrate activation and methyl-transfer limitation | The pH and lysine-substitution patterns are used to propose an H120 base and R121 stabilization role. They do not independently measure residue-specific protonation or eliminate local folding and binding effects. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Four 7-position substrate-analogue comparisons | supports_proposed_substrate_activation | Proposed substrate activation and methyl-transfer limitation | The four negative 7-position analogues support the source 7-OH-dependent activation model under these conditions; they do not establish the same chemistry for mature antibiotic or every NovO substrate. | https://strathprints.strath.ac.uk/60153/1/Sadler_etal_ACSCB_2017_C_methylation_of_coumarin_scaffolds_by_NovO.pdf
|
| Deposited native NovO sequence reference | is_native_reference_for | Sadler NovO-His6 WT kinetic comparator | UniProtKB:Q9L9F3 is the deposited native sequence reference for Sadler NovO-His6 WT kinetic comparator (node 'wt'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F3 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 14 edges
Native NovP identity, recombinant preparation and explicitly database-annotated precursor methylation. Native sequence, DNA construct and tagged protein remain distinct. Review #558. The 2010 page locators refer to the 30-page author manuscript, not journal pagination. The cathomycin owner retains its unspecified sugar stereocentre; no exact merge with fully specified novobiocin or new mature-product synthesis claim is made.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novP locus from NCIMB 9219 | The owner links BGC0000834.5 and AF170880.4. novP is 18275..19063(+) with a 789-nt CDS including the stop codon. This is a locus association, not independent proof of the owner sugar stereochemistry. | https://mibig.secondarymetabolites.org/go/BGC0000834.5 |
| Deposited novP locus from NCIMB 9219 | encodes_native_protein | Deposited native NovP sequence reference | Table-11 CDS-aware translation matches AAF67509.1 and all 262 residues of Q9L9F2. Current GenBank source is S. niveus NCIMB 9219, taxon 193462. This accession identifies the native sequence reference, not tagged protein. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited native NovP sequence reference | has_unresolved_sample_attribution | NovP source-taxon and historical strain disagreement | UniProt names S. niveus with S. spheroides as common name. The 2010 paper names S. spheroides; 2WK1 mmCIF retains common S. spheroides, scientific S. caeruleus, taxon 195949 and NCIB 11891. The current RCSB summary renders Actinoalloteichus caeruleus. Identical sequence does not resolve historical sample provenance or identify NCIB 11891 as NCIMB 9219; no owner producer field is changed. | https://www.uniprot.org/uniprotkb/Q9L9F2 |
| Deposited native NovP sequence reference | is_sequence_reference_for | pET28a NovP N-terminal His-tag DNA construct | The 2007 methods report genomic PCR and cloning into pET28a. The exact deposited protein core matches Q9L9F2; this comparison does not independently sequence the full experimental plasmid or settle the source-strain conflict. | DOI:10.1107/S1744309107008287 |
| pET28a NovP N-terminal His-tag DNA construct | encodes_engineered_protein | Gomez Garcia N-terminally tagged NovP preparation | The 282-aa deposited construct consists of MGSSHHHHHHSSGLVPRGSH (20 residues) followed by the exact 262-aa native core. The N-terminal tag was not cleaved before crystallization. Unmodeled tag coordinates are not evidence for proteolytic removal, and no native accession is assigned to this engineered preparation. | DOI:10.1016/j.jmb.2009.10.045 |
| E. coli BL21(DE3) NovP expression host | expressed | Gomez Garcia N-terminally tagged NovP preparation | The 2007 protocol uses BL21(DE3), 1 L LB with 50 mg kanamycin, 24 h at 298 K without IPTG induction, French-press lysis and nickel affinity purification. This is recombinant production, not native abundance. | DOI:10.1107/S1744309107008287
|
| Gomez Garcia N-terminally tagged NovP preparation | has_reported_preparation_readout | Reported NovP purification yield and purity | The 2007 source reports about 20 mg purified NovP per L culture and >95% purity by SDS-PAGE. Yield and gel purity are not an activity measurement, an active-fraction estimate or a natural-product titer. | DOI:10.1107/S1744309107008287
|
| Gomez Garcia N-terminally tagged NovP preparation | was_used_in | SAH-containing NovP crystallization experiment | The 2007 source reports approximately 10 mg/mL protein, 1 mM SAH, 291 K and optimized 2 M ammonium sulfate/100 mM HEPES pH 7 precipitant. No apo conditions were found in that screen; this does not establish that apo protein cannot crystallize under other conditions. | DOI:10.1107/S1744309107008287 |
| S-Adenosyl-L-homocysteine coproduct and crystallization ligand | was_supplied_to | SAH-containing NovP crystallization experiment | SAH was supplied as a ligand; the crystals are not evidence that the crystallization sample catalyzed methylation or produced SAH in situ. | DOI:10.1107/S1744309107008287 |
| Deposited native NovP sequence reference | has_database_annotated_function | Database-annotated noviose 4-O-methylation | UniProt annotates SAM-dependent noviose 4-O-methylation as the penultimate novobiocin step, citing the 2004 biochemical studies. Those complete primary assays were not inspected here; this edge remains a database assertion. Introductory kinetic constants in the 2010 paper are not curated as newly measured experiments. | https://www.uniprot.org/uniprotkb/Q9L9F2
|
| Desmethyldescarbamoylnovobiocin acceptor | is_annotated_acceptor_for | Database-annotated noviose 4-O-methylation | The database reaction names desmethyldescarbamoylnovobiocin, not mature cathomycin, as methyl acceptor. No uninspected exact substrate CURIE is assigned. | https://www.uniprot.org/uniprotkb/Q9L9F2
|
| S-Adenosyl-L-methionine donor | is_annotated_donor_for | Database-annotated noviose 4-O-methylation | SAM supplies the methyl group in the annotated reaction; this is not a donor-loading measurement from the crystallization study. | https://www.uniprot.org/uniprotkb/Q9L9F2
|
| Database-annotated noviose 4-O-methylation | has_annotated_product | Descarbamoylnovobiocin precursor product | The methylated product still lacks the later carbamoyl substituent. This edge does not assert mature antibiotic synthesis by NovP alone. | https://www.uniprot.org/uniprotkb/Q9L9F2
|
| Database-annotated noviose 4-O-methylation | has_annotated_coproduct | S-Adenosyl-L-homocysteine coproduct and crystallization ligand | The annotated balanced reaction also releases SAH and H+. No stoichiometric assay or direct proton-transfer observation is inferred. | https://www.uniprot.org/uniprotkb/Q9L9F2
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 8 nodes, 9 edges
Separate author/PISA dimer, dimer-like 2010 SEC and monomer-like 2007 DLS evidence. None uniquely establishes native-cell stoichiometry. Review #558. The 2010 page locators refer to the 30-page author manuscript, not journal pagination. The cathomycin owner retains its unspecified sugar stereocentre; no exact merge with fully specified novobiocin or new mature-product synthesis claim is made.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Gomez Garcia N-terminally tagged NovP preparation | was_crystallized_for | 2WK1 NovP-SAH crystallographic model | 2WK1 has one protein chain in the asymmetric unit. A two-chain biological assembly is not two independently observed asymmetric-unit chains. | DOI:10.1016/j.jmb.2009.10.045 |
| 2WK1 NovP-SAH crystallographic model | supports_proposed_assembly | Author/PISA symmetry-generated NovP dimer | The authors and PISA assign a dimer generated by operations x,y,z and -x,y,-z+1. The paper reports approximately 856 A^2 buried surface per subunit, about 8%, mainly involving helices alpha1 and alpha7. This is crystal-interface analysis, not direct native-cell assembly measurement. | DOI:10.1016/j.jmb.2009.10.045 |
| Gomez Garcia N-terminally tagged NovP preparation | has_measured_solution_size_in | 2010 NovP calibrated size-exclusion result | After nickel affinity and buffer exchange, approximately 2 mL at 10 mg/mL was loaded on Superdex 75 16/60 in 50 mM Tris-HCl, 200 mM NaCl pH 8.0, at 1 mL/min. Standards were albumin 67, ovalbumin 43, chymotrypsinogen A 25, ribonuclease A 13.7 and dextran blue 2000 kDa. The main fraction had apparent size about 66 kDa; no monomer peak was reported. The manuscript does not display a SEC chromatogram. | DOI:10.1016/j.jmb.2009.10.045
|
| 2010 NovP calibrated size-exclusion result | supports_dimer_like_size_for | Author/PISA symmetry-generated NovP dimer | The reported approximately 66-kDa apparent mass is near the source 64.2-kDa expected tagged dimer. SEC is hydrodynamic evidence for that preparation, not a measured association constant or native in-cell stoichiometry. | DOI:10.1016/j.jmb.2009.10.045
|
| Gomez Garcia N-terminally tagged NovP preparation | has_earlier_solution_size_estimate | 2007 NovP dynamic-light-scattering result | The 2007 DLS experiment at 293 K used at least 15 measurements, giving a monomodal distribution, 17.1% polydispersity and apparent mass 32.8 kDa, compared with calculated 32.1-kDa His-tagged monomer. It is a different method/preparation context from the later SEC result. | DOI:10.1107/S1744309107008287
|
| 2007 NovP dynamic-light-scattering result | limits_unique_stoichiometry_claim | Preparation-specific NovP stoichiometry limits | Retain the monomer-like DLS estimate rather than silently converting it to the later dimer assignment. These data do not by themselves resolve concentration dependence, shape effects or a monomer-dimer equilibrium. | DOI:10.1107/S1744309107008287 |
| Author/PISA symmetry-generated NovP dimer | has_context_limit | Preparation-specific NovP stoichiometry limits | The structural/PISA model and SEC support a tagged-protein dimer under the studied conditions; the earlier DLS estimate remains distinct. No unique native-cell stoichiometry is asserted. | DOI:10.1016/j.jmb.2009.10.045 |
| Author/PISA symmetry-generated NovP dimer | does_not_establish_assembly_of | Unverified transfer of NovP interface to homologs | The paper predicts related CouP/CloP interfaces from conservation and is less certain about TylF. NovP coordinates do not experimentally demonstrate an identical dimer interface in these other proteins. | DOI:10.1016/j.jmb.2009.10.045
|
| Deposited native NovP sequence reference | is_native_reference_for | Gomez Garcia N-terminally tagged NovP preparation | UniProtKB:Q9L9F2 is the deposited native sequence reference for Gomez Garcia N-terminally tagged NovP preparation (node 'wt'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F2 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 10 edges
Observed SAH contacts and structural limits, with independent coordinate checks kept separate from the printed Asp128 label and from functional inference. Review #558. The 2010 page locators refer to the 30-page author manuscript, not journal pagination. The cathomycin owner retains its unspecified sugar stereocentre; no exact merge with fully specified novobiocin or new mature-product synthesis claim is made.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Gomez Garcia N-terminally tagged NovP preparation | was_crystallized_for | 2WK1 NovP-SAH crystallographic model | The 1.4-A SAH-bound structure represents the tagged recombinant protein, not untagged native NovP or a substrate-containing catalytic intermediate. | DOI:10.1016/j.jmb.2009.10.045 |
| 2WK1 NovP-SAH crystallographic model | contains_observed_ligand | Observed S-adenosyl-L-homocysteine ligand | The deposited SAH has 26 heavy atoms. Figure 6 shows its simulated-annealing omit map at 6 sigma and describes Phe178/adenine stacking. Occupancy and packing do not provide a dissociation constant or prove tight binding in solution. | DOI:10.1016/j.jmb.2009.10.045 |
| Observed S-adenosyl-L-homocysteine ligand | has_independently_checked_contacts | Observed SAH polar-contact geometry | native 64 OG1--SAH OXT 2.593 A; native 65 N--SAH O 2.855 A; native 92 OE2--SAH N 2.721 A; native 94 O--SAH N 2.901 A; native 96 N--SAH O3' 3.120 A; native 122 OD1--SAH O2' 2.752 A; native 122 OD2--SAH O3' 2.643 A; native 196 OD2--SAH N 2.755 A; native 202 OG--SAH N7 2.843 A; native 202 OG--SAH N6 2.964 A. These ten selected coordinate distances support geometrical contacts, not hydrogen-bond energies or proton transfer. Residue numbers are native/auth_seq_id, not label_seq_id (which includes the +20 tag offset). | DOI:10.1016/j.jmb.2009.10.045 |
| 2WK1 NovP-SAH crystallographic model | has_unresolved_regions | Unresolved NovP residues and illustrative loop model | 242 residues are modeled: native 14..38 and 46..262. The missing 40 comprise tag positions -19..0, native 1..13 and 39..45. The cyan loop 39..45 in Figure 2 is an energetically favorable illustrative model, not experimentally resolved coordinates. Asp14/Arg159/Lys241/Arg262 lack side-chain atoms; alternate conformations occur at Met22, Asp165, Thr189, Met226, Glu237, Ile249 and Arg260. | DOI:10.1016/j.jmb.2009.10.045 |
| 2WK1 NovP-SAH crystallographic model | has_source_specific_quality_values | Source-specific NovP refinement statistics | The author manuscript reports Rwork 14.5% and Rfree 16.2%; current deposited refinement values are 0.145 and 0.163. Preserve the source-specific difference rather than silently harmonizing it. The 2007 maximum 1.35-A collection and reprocessed 1.40-A dataset are different reports, not two resolutions for the final atomic model. | DOI:10.1016/j.jmb.2009.10.045 |
| 2WK1 NovP-SAH crystallographic model | contains_observed_region | NovP residues 126-151 lid region | Residues 126..151 form the helical lid over mostly buried SAH. The paper gives lid main/side-chain B factors 20.5/24.7 A^2 versus 14.0/17.7 for the rest of the protein. Table 2 whole-protein values 14.6/18.1 use another denominator. Static B factors are not direct measurements of gating trajectories, exchange rates or rate-limiting lid motion. | DOI:10.1016/j.jmb.2009.10.045
|
| NovP residues 126-151 lid region | has_unresolved_source_residue_label | Printed Asp128 versus native Pro128 and Asp138 geometry | Page 5 first names Gly126/Pro128 and later prints Asp128 as contacting Tyr200/Glu201. Native 128 is Pro, whereas 138 is Asp. Independent 2WK1 distances are Asp138 OD1--Tyr200 N 2.763 A and Asp138 OD2--Glu201 N 3.000 A. These support a possible residue-number error, not an authorized correction of the source or direct proof of a lid-locking mechanism. | DOI:10.1016/j.jmb.2009.10.045 |
| 2WK1 NovP-SAH crystallographic model | contains_observed_disulfide | Observed Cys228-Cys231 disulfide geometry | Cys228 and Cys231 have deposited SG separation 2.167 A and an explicit disulfide record. The authors consider prolonged aerobic exposure during up-to-one-month crystal growth as a possible artifact. No physiological redox role, obligatory catalytic function or experimentally demonstrated activity loss caused by this bond is inferred. | DOI:10.1016/j.jmb.2009.10.045 |
| 2WK1 NovP-SAH crystallographic model | has_observed_coordinate_inventory | Observed ligands and absent modeled catalytic partners | The asymmetric unit contains 1965 protein atoms, 26 SAH atoms, two sulfates, two ethylene glycol molecules and 242 waters. Deposited coordinates contain no metal, SAM or DDN. Reported MgCl2 soaking and co-crystallization also gave no metal density, but those trials are data-not-shown and are not separately displayed structures. | https://files.rcsb.org/download/2WK1.cif |
| Deposited native NovP sequence reference | is_native_reference_for | Gomez Garcia N-terminally tagged NovP preparation | UniProtKB:Q9L9F2 is the deposited native sequence reference for Gomez Garcia N-terminally tagged NovP preparation (node 'wt'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F2 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 18 nodes, 17 edges
NovP substrate docking and structural hypotheses are explicitly computational, not observed catalytic complexes, engineered-mutant results or protein interactions. Review #558. The 2010 page locators refer to the 30-page author manuscript, not journal pagination. The cathomycin owner retains its unspecified sugar stereocentre; no exact merge with fully specified novobiocin or new mature-product synthesis claim is made.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| 2WK1 NovP-SAH crystallographic model | was_template_for_docking_receptor | Manually prepared NovP-SAM-Mg docking receptor | The authors removed solvent, replaced SAH with SAM, inserted Mg2+ using homolog structures and manually adjusted Asp196/Asp223/Asp224. They performed 300 CNS maximum-likelihood minimization cycles with Wa set to zero. This is an engineered computational receptor, not an experimentally observed NovP-SAM-Mg complex. | DOI:10.1016/j.jmb.2009.10.045
|
| Modeled S-adenosyl-L-methionine donor | was_modeled_into | Manually prepared NovP-SAM-Mg docking receptor | SAM replaces the observed SAH before docking; no experimental SAM electron density is supplied by 2WK1. | DOI:10.1016/j.jmb.2009.10.045
|
| Modeled magnesium ion | was_modeled_into | Manually prepared NovP-SAM-Mg docking receptor | Metal placement and Asp conformations are assumptions guided by homologs, not an observed catalytic metal or measured metal affinity. | DOI:10.1016/j.jmb.2009.10.045
|
| Manually prepared NovP-SAM-Mg docking receptor | was_used_in | AutoDock4 NovP substrate-pose search | AutoDock4 treated the receptor as rigid except Asp198/Trp58 side chains. The search used a 26-A cube, 0.375-A grid, 100 runs, population 150, 25000000 energy evaluations, 27000 generations, one surviving conformation per generation, 300 local iterations and 2-A clustering tolerance. | DOI:10.1016/j.jmb.2009.10.045
|
| Docked desmethyldescarbamoylnovobiocin | was_flexible_ligand_in | AutoDock4 NovP substrate-pose search | DDN had a random starting position and rotatable single bonds. The authors defined a productive pose as placing noviose 4-OH within 4 A of the SAM methyl group. This geometric filter does not demonstrate catalysis. | DOI:10.1016/j.jmb.2009.10.045
|
| AutoDock4 NovP substrate-pose search | has_pre_deprotonation_result | Pre-deprotonation docking scenario | With protonated noviose 4-OH and deprotonated Asp198, 3 of 100 poses passed the geometric filter; best docking energy was printed as -8.68 kJ/mol. These are source model units/counts, not measured turnover, a 3% chemical conversion or experimental binding free energy. | DOI:10.1016/j.jmb.2009.10.045
|
| AutoDock4 NovP substrate-pose search | has_post_deprotonation_result | Post-deprotonation docking scenario | With deprotonated noviose 4-OH and protonated Asp198, 98 of 100 poses passed the filter; best docking energy was printed as -15.88 kJ/mol. Proton transfer is assumed in this scenario, not observed by the simulation or an assay. | DOI:10.1016/j.jmb.2009.10.045
|
| Post-deprotonation docking scenario | was_clustered_into | Twelve retained post-deprotonation pose clusters | The 98 retained poses formed 12 clusters. Their lowest-energy representatives place ring C similarly, ring B in roughly four orientational groups and ring A more variably outside the channel. This is not a new experimental substrate-tolerance panel. The lid is removed graphically in Figure 9, not deleted from an assayed protein. | DOI:10.1016/j.jmb.2009.10.045
|
| Manually prepared NovP-SAM-Mg docking receptor | assumes_metal_binding_by | Proposed Asp196/Asp223/Asp224 metal-binding roles | Asp196/Asp223/Asp224 are proposed ligands from geometry/conservation and homolog comparisons. No catalytic metal is observed in 2WK1 and no metal-site mutant panel is reported in this paper. | DOI:10.1016/j.jmb.2009.10.045
|
| Manually prepared NovP-SAM-Mg docking receptor | assumes_general_base_role_for | Proposed Asp198 general-base role | Asp198 is proposed to deprotonate noviose 4-OH. Its orientation and homolog comparisons motivate the model, but there is no reported D198 mutant assay or directly measured proton-transfer step here. | DOI:10.1016/j.jmb.2009.10.045
|
| Twelve retained post-deprotonation pose clusters | suggest_contact_with | Proposed Trp58 sugar-ring contact | The Trp58 side chain was flexible in the docking and is proposed to contact the noviose ring oxygen. No substrate-bound experimental NovP structure establishes this contact. | DOI:10.1016/j.jmb.2009.10.045
|
| Twelve retained post-deprotonation pose clusters | suggest_contact_with | Proposed Arg146 aminocoumarin contact | Arg146 points toward ring B in the models and is proposed to recognize its C8 substituent. This is neither a demonstrated residue-specific binding energy nor an assayed R146 mutant. | DOI:10.1016/j.jmb.2009.10.045
|
| 2WK1 NovP-SAH crystallographic model | motivates_binding_order_hypothesis | Proposed SAM-first and SAH-last binding order | Buried SAH/lid geometry and comparison to TylF motivate SAM binding before DDN and SAH leaving last. This is not a directly measured NovP ordered Bi Bi mechanism or a kinetic inhibition experiment. | DOI:10.1016/j.jmb.2009.10.045
|
| 2WK1 NovP-SAH crystallographic model | is_discussed_with_undisplayed_assay_report | Undisplayed personal-communication metal tests | The paper relays Freel Meyers personal communication: 10 mM MgCl2 or MnCl2 had no measurable effect, and 10 mM CaCl2 reduced product. No underlying data, quantitative effect size, replication or assay recipe is displayed. The source proposes competition with trace preferred metals, but this is not an independently inspected metal-dependence or inhibition experiment. | DOI:10.1016/j.jmb.2009.10.045
|
| 2WK1 NovP-SAH crystallographic model | motivates_unverified_interaction_hypothesis | Untested NovP-NovM/NovN interaction proposal | The authors speculate that NovM or NovN interactions could assist lid opening or channel intermediates, explicitly stating that there is no evidence either way. No binding edge, observed complex, stoichiometry or functional interaction is asserted here. | DOI:10.1016/j.jmb.2009.10.045
|
| 2WK1 NovP-SAH crystallographic model | motivates_untested_deletion_proposal | Suggested untested deletion of lid residues 126-151 | Complete removal of residues 126..151 is proposed for future engineering. The paper does not report constructing, purifying or assaying this deletion; graphical lid omission does not create a tested mutant protein entry. | DOI:10.1016/j.jmb.2009.10.045
|
| 2WK1 NovP-SAH crystallographic model | has_reported_unsuccessful_extension | Unsuccessful reported additional ligand-complex trials | Trials used descarbamoylnovobiocin or methylrhamnoside, with SAM/SAH and with/without MgCl2, but yielded no successful complexes and are data-not-shown. DDN was insufficient for these experiments. Failure to obtain crystals is not evidence that the enzyme cannot bind these molecules in solution. | DOI:10.1016/j.jmb.2009.10.045
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 11 edges
NovN deposited sequence versions, literal primers and the Xu His6 preparation. Review #560. Xu locators are PDF pages, nineteen higher than printed main-text pages. The owner retains its unspecified sugar stereocentre and SEEDED status; source-assigned novobiocin is not an exact structure-key merge with cathomycin. Native reference, engineered protein and DNA construct are separate entities.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novN locus from NCIMB 9219 | The owner links BGC0000834.5 and AF170880.4. The current novN CDS is 15274..17307(+), 2034 nt including stop. This is a locus association, not independent proof of the owner sugar stereochemistry. | https://mibig.secondarymetabolites.org/go/BGC0000834.5 |
| Deposited novN locus from NCIMB 9219 | encodes_native_reference | Current deposited native NovN sequence reference | CDS-aware table-11 translation exactly matches AAF67507.2 and all 677 residues of reviewed Q9L9F4. GenBank names S. niveus NCIMB 9219; UniProt names S. niveus with common name S. spheroides, taxon 193462. The accession is not assigned to an unsequenced engineered preparation. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novN locus from NCIMB 9219 | has_earlier_deposited_translation | Historical deposited NovN sequence version | AF170880.2 has AAF67507.1, also 677 aa, with LRRN at residues 123..126 where current AAF67507.2/Q9L9F4 has APPD. AF170880.3 already matches the current protein. In .2/.3 novN is 15222..17255; .4 coordinates are shifted +52. Deposit revisions are not independently proven biological alleles. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.2 |
| Historical deposited NovN sequence version | constrains_sample_identification | NovN historical sample and sequence-version limits | The thesis names S. spheroides and an AF170880-derived cloning source, but no complete experimental pXHNEG sequence was obtained. Current database identity does not establish which deposited 123..126 sequence was present in that assayed sample or resolve historical strain labels. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1 |
| Deposited novN locus from NCIMB 9219 | is_reported_insert_source_for | Xu pXHNEG N-terminal His6 NovN DNA construct | Xu cloned the PCR product through pGEM-T (pXHNET) and pRSET-B (pXHNER; BclI/BamHI-compatible plus HindIII ends), then fused pXHNER and pGM9 at HindIII to make pXHNEG. Figure 15 gives 11298 bp. The Table 14 2.1-kb fragment label accompanies 2034-nt CDS coordinates; CDS length is not the complete cloning-fragment length. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu pXHNEG N-terminal His6 NovN DNA construct | has_independently_checked_primer_model | Literal novN primer model and coordinate discrepancy | Literal primers GTGCTCGCTGATCAGAACGACATG and AAGGGAAGCTTTACGGCCGAC have unique best full-length genomic matches with three mismatches each. The forward span is 15201..15224 in .2/.3, not printed 15198..15221; reverse-complement span 17285..17307 agrees. Current coordinates shift +52, which does not explain the separate three-base discrepancy. The modeled PCR is 2107 bp; BclI/HindIII cut coordinates differ by 2088 nt. Native core is retained in each version-specific model. This is not a sequenced full plasmid or independently determined tag junction. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1 |
| Xu pXHNEG N-terminal His6 NovN DNA construct | encodes_engineered_protein | Xu N-terminal His6 NovN preparation | Xu reports N-terminal His6 NovN, separate from the later C-terminal LE-His8 protein. The native accession and historical deposited variant are not identities for this fusion. No complete vector-derived amino-acid sequence or active-protein fraction is inferred. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| S. lividans T7 NovN expression host | expressed | Xu N-terminal His6 NovN preparation | The TK23-derived S. lividans T7 host supplies thiostrepton-induced T7 polymerase. YEME with 10 ug/mL kanamycin was grown at 28 C and 180 rpm for 48 h; 1 mL inoculated 100 mL fresh medium, with 25 ug/mL thiostrepton for 24 h. Lowering growth to 20 C did not substantially improve soluble NovN. These are heterologous protein-preparation conditions. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
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| Xu N-terminal His6 NovN preparation | has_reported_preparation_yield | Xu NovN protein yield and solubility readout | The source estimates 6.7 mg NovN/g fresh cells before purification, much insoluble, and about 16 ug purified NovN/g fresh cells from SDS-PAGE. Ni-NTA purification and NAP-10 desalting into 0.2 M Tris-HCl pH 8 precede assays. Bradford/BSA is the general protein concentration method. These mass-per-biomass yields are not mg/L culture, active-enzyme concentrations or antibiotic titers. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu N-terminal His6 NovN preparation | has_reported_gel_profile | Xu NovN SDS-PAGE bands and proposed degradation | Figure 16 distinguishes pre/post induction, soluble fraction and affinity eluate. A roughly 78-kDa band agrees with the source-calculated 78.5 kDa; an additional 76-kDa band is proposed to reflect degradation. No cleavage site, fragment sequence or activity of that band was established. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
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| NovN historical sample and sequence-version limits | limits_native_accession_assignment_to | Xu N-terminal His6 NovN preparation | Keep this experimentally active preparation ungrounded as an exact protein entity. A supported novN gene assignment is retained without inventing a verified native core, complete N-terminal tag or historical sample identity. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 16 nodes, 16 edges
Xu purified NovN carbamoylation, conditional kinetics and bounded negative tests. Review #560. Xu locators are PDF pages, nineteen higher than printed main-text pages. The owner retains its unspecified sugar stereocentre and SEEDED status; source-assigned novobiocin is not an exact structure-key merge with cathomycin. Native reference, engineered protein and DNA construct are separate entities.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Xu N-terminal His6 NovN preparation | catalyzes | Xu NovN carbamoylation endpoint assay | The standard assay uses 100 uL, 0.2 M Tris-HCl pH 8, 1 mM acceptor, 5 mM carbamoyl-phosphate dilithium, 2 mM ATP, 2 mM Mg and 0.4 uM NovN at 30 C, quenched with 5 uL 1.5 M trichloroacetic acid. Overnight endpoint/preparative incubations are not the five-minute kinetic assay. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Descarbamoylnovobiocin acceptor | is_substrate_for | Xu NovN carbamoylation endpoint assay | Descarbamoylnovobiocin is the genuine acceptor named by the source. Materials identify Pharmacia & Upjohn as supplier. No exact chemical CURIE or new resolution of the owner sugar stereochemistry is inferred. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Carbamoyl-phosphate dilithium donor | is_carbamoyl_donor_for | Xu NovN carbamoylation endpoint assay | Carbamoyl phosphate is supplied at 5 mM. The experiment supports carbamoyl transfer under the stated conditions, not independent quantitation of phosphate release or donor binding. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu NovN carbamoylation endpoint assay | forms_source_assigned_product | HPLC-assigned novobiocin assay product | Figure 17 shows the source-assigned novobiocin signal. Products were extracted twice with 500 uL ethyl acetate and separated on Nucleosil RP120-5 with 70-100% methanol in 1% formic acid over 25 min, detected at 325 nm. The methods name clorobiocin and descarbamoylnovobiocin standards; this edge does not invent an independently inspected novobiocin standard or establish exact-key equivalence to the owner. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Heat-denatured NovN control | is_negative_control_for | Xu NovN carbamoylation endpoint assay | The methods specify heat-denatured enzyme as a negative control. This is the stated control design, not a separately quantified detection limit or a measured fraction of active enzyme. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu NovN carbamoylation endpoint assay | has_reported_atp_dependence | ATP omission and supplementation readout | ATP omission is reported to eliminate detectable enzymatic activity; no numerical detection threshold is supplied. The stated optimum is 2 mM ATP. This does not establish stoichiometric ATP consumption or an ATP-binding constant. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu NovN carbamoylation endpoint assay | has_reported_metal_dependence | Added Mg/Mn dependence and residual activity | With ATP but without added Mg2+ or Mn2+, the source reports 1% activity, not zero. The stated optimum Mg2+ concentration is 2 mM. This is not an exhaustive metal-specificity panel or evidence that the later deposited Fe is a physiological cofactor. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu N-terminal His6 NovN preparation | was_measured_in | Xu five-minute NovN kinetic experiment | Kinetics used 37.5 nM NovN, 5 mM carbamoyl phosphate, 2.5-40 uM acceptor and five-minute incubations under the reported assay protocol. Assays were duplicate and evaluated by Lineweaver-Burk plots. Figure error bars are not defined as SD or SE. The source reports linear product formation up to 0.5 uM enzyme and 90 min in preliminary tests. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Descarbamoylnovobiocin acceptor | conditions | Descarbamoylnovobiocin-conditioned NovN kinetic fit | For descarbamoylnovobiocin the thesis reports Km 2.4 uM and kcat 2.4 min^-1. These preparation- and condition-specific values are not binding affinity, native flux or the separate Freel Meyers values of 4.6 uM and 4.1 min^-1 relayed by UniProt. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1 |
| Xu five-minute NovN kinetic experiment | yields_reported_fit | Descarbamoylnovobiocin-conditioned NovN kinetic fit | The reported Lineweaver-Burk fit applies to the genuine acceptor; no uncertainty interval or active-fraction correction is supplied. Do not transfer the 0.4-uM overnight enzyme loading to this fit. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| ATP omission and supplementation readout | motivates_source_hypothesis | Proposed Mg-ATP allosteric activation | The discussion proposes Mg-ATP as an allosteric activator. This is a source hypothesis, not a measured allosteric site, ATP occupancy, proton-transfer mechanism or a solved catalytic intermediate. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu N-terminal His6 NovN preparation | was_tested_in | Crude coumermycin D extract test | Coumermycin D was supplied as crude mutant extract, not pure compound. Its nominal 0.1 mM concentration was calculated as coumermycin A1. The overnight in-vitro test therefore has matrix and concentration limits not shared by the purified acceptor assays. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Crude coumermycin D extract test | gave_bounded_negative_readout | No expected in-vitro coumermycin D product detected | The expected carbamoylated product was not detected in this in-vitro extract assay. Do not encode universal nonacceptance of pure coumermycin D: the separate heterologous experiment reports a minor bis-carbamoylated product. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu N-terminal His6 NovN preparation | was_tested_in | Acetyl-phosphate donor substitution test | The alternative-donor experiment supplied 5 mM acetyl phosphate instead of carbamoyl phosphate with the genuine acceptor overnight. A carbamoyl-phosphate positive and acceptor-only negative control bound interpretation of this particular substitution. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Acetyl-phosphate donor substitution test | gave_bounded_negative_readout | No expected acetyl-phosphate-derived product detected | No expected acetyl-phosphate-derived product was detected under these conditions. This does not exclude all alternative donors, all enzyme loadings or activity below an unspecified detection threshold. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Deposited native NovN sequence reference | is_native_reference_for | Xu N-terminal His6 NovN preparation | UniProtKB:Q9L9F4 is the deposited native sequence reference for Xu N-terminal His6 NovN preparation (node 'wt'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F4 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 17 nodes, 18 edges
Five source-assigned NovN analogue products and two separate analogue kinetic fits. Review #560. Xu locators are PDF pages, nineteen higher than printed main-text pages. The owner retains its unspecified sugar stereocentre and SEEDED status; source-assigned novobiocin is not an exact structure-key merge with cathomycin. Native reference, engineered protein and DNA construct are separate entities.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Xu N-terminal His6 NovN preparation | catalyzes | Five-analogue NovN overnight carbamoylation panel | The five analogue acceptors 104, 105, 107, 108 and 283 were incubated overnight with carbamoyl phosphate under the endpoint protocol (1 mM acceptor, 5 mM donor, 2 mM ATP/Mg, 0.4 uM enzyme, 30 C). Product assignments use HPLC, NMR and negative FAB-MS. These are source structural assignments, not new independent reassignments. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Five-analogue NovN overnight carbamoylation panel | has_reported_aggregate_conversion | Mean conversion for four non-104 acceptors | The source gives 87% overnight conversion for 104 and about 54% as the average for the other four acceptors. The latter is not four individual measurements, a rate or a common uncertainty estimate. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu N-terminal His6 NovN preparation | was_measured_in | Xu five-minute analogue kinetic comparison | The separate kinetic comparison uses 37.5 nM NovN, 5 mM donor, 2.5-40 uM acceptor and five-minute incubations, duplicate assays and Lineweaver-Burk analysis. It is not the overnight conversion panel. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Novclobiocin 104 acceptor | is_tested_acceptor_for | Five-analogue NovN overnight carbamoylation panel | The source names novclobiocin 104 as this panel acceptor; no exact chemical CURIE or equivalence to the owner is assigned. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Five-analogue NovN overnight carbamoylation panel | forms_source_assigned_product | Source-assigned novclobiocin 114 | For the 104->114 reaction, the source assigns 3-double-prime carbamoylation from H-3 double-prime shifting 4.18->5.37 ppm and a +43-Da mass change. Product negative FAB [M-H]- is reported as m/z 631, a source integer assignment rather than an exact mass. This is the 87% overnight conversion row; no error estimate is given. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Novclobiocin 105 acceptor | is_tested_acceptor_for | Five-analogue NovN overnight carbamoylation panel | The source names novclobiocin 105 as this panel acceptor; no exact chemical CURIE or equivalence to the owner is assigned. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Five-analogue NovN overnight carbamoylation panel | forms_source_assigned_product | Source-assigned novclobiocin 115 | For the 105->115 reaction, the source assigns 3-double-prime carbamoylation from H-3 double-prime shifting 4.12->5.27 ppm and a +43-Da mass change. Product negative FAB [M-H]- is reported as m/z 617, a source integer assignment rather than an exact mass. Table 22 gives the acceptor H-3'' coupling as 3.1 Hz but Appendix I gives 3.3 Hz; Appendix I also lists C-8'' at 62.3 ppm then calls it undetectable. These conflicts are retained, not used to invent a revised structure. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Novclobiocin 107 acceptor | is_tested_acceptor_for | Five-analogue NovN overnight carbamoylation panel | The source names novclobiocin 107 as this panel acceptor; no exact chemical CURIE or equivalence to the owner is assigned. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Five-analogue NovN overnight carbamoylation panel | forms_source_assigned_product | Source-assigned novclobiocin 117 | For the 107->117 reaction, the source assigns 3-double-prime carbamoylation from H-3 double-prime shifting 4.09->5.25 ppm and a +43-Da mass change. Product negative FAB [M-H]- is reported as m/z 597, a source integer assignment rather than an exact mass. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Novclobiocin 108 acceptor | is_tested_acceptor_for | Five-analogue NovN overnight carbamoylation panel | The source names novclobiocin 108 as this panel acceptor; no exact chemical CURIE or equivalence to the owner is assigned. Figure 19 panel D labels 108 although the caption omits its number. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Five-analogue NovN overnight carbamoylation panel | forms_source_assigned_product | Source-assigned novclobiocin 118 | For the 108->118 reaction, the source assigns 3-double-prime carbamoylation from H-3 double-prime shifting 4.03->5.15 ppm and a +43-Da mass change. Product negative FAB [M-H]- is reported as m/z 583, a source integer assignment rather than an exact mass. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Novclobiocin 283 acceptor | is_tested_acceptor_for | Five-analogue NovN overnight carbamoylation panel | The source names novclobiocin 283 as this panel acceptor; no exact chemical CURIE or equivalence to the owner is assigned. The source attributes 283 supply to an earlier 3-bromo-4-hydroxybenzoic-acid feeding experiment; this is not a new feeding experiment in the NovN assay. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Five-analogue NovN overnight carbamoylation panel | forms_source_assigned_product | Source-assigned novclobiocin 284 | For the 283->284 reaction, the source assigns 3-double-prime carbamoylation from H-3 double-prime shifting 4.19->5.37 ppm and a +43-Da mass change. Product negative FAB [M-H]- is reported as m/z 643, a source integer assignment rather than an exact mass. Product H-4'' is 3.56 ppm in Table 22 and 3.55 in Appendix I; the table contains the 283 precursor H-3 value but Appendix I lists only 284. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Novclobiocin 104 acceptor | conditions | Novclobiocin 104-conditioned NovN kinetic fit | The source reports Km 1.9 uM and kcat 1.32 min^-1 for novclobiocin 104; no uncertainty interval is stated. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu five-minute analogue kinetic comparison | yields_reported_fit | Novclobiocin 104-conditioned NovN kinetic fit | The value is a preparation-specific kinetic fit, not the overnight endpoint percentage or a native-pathway flux. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Novclobiocin 105 acceptor | conditions | Novclobiocin 105-conditioned NovN kinetic fit | The source reports Km 14.3 uM and kcat 1.53 min^-1 for novclobiocin 105; no uncertainty interval is stated. The source calls 14.3 versus the genuine acceptor 2.4 uM a fivefold difference; the literal ratio is approximately 5.96. Preserve the reported values and distinguish the source wording. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu five-minute analogue kinetic comparison | yields_reported_fit | Novclobiocin 105-conditioned NovN kinetic fit | The value is a preparation-specific kinetic fit, not the overnight endpoint percentage or a native-pathway flux. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Deposited native NovN sequence reference | is_native_reference_for | Xu N-terminal His6 NovN preparation | UniProtKB:Q9L9F4 is the deposited native sequence reference for Xu N-terminal His6 NovN preparation (node 'wt'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F4 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 12 nodes, 12 edges
pXHN construct evidence and source-assigned heterologous coumermycin analogue production. Review #560. Xu locators are PDF pages, nineteen higher than printed main-text pages. The owner retains its unspecified sugar stereocentre and SEEDED status; source-assigned novobiocin is not an exact structure-key merge with cathomycin. Native reference, engineered protein and DNA construct are separate entities.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| novN DNA insert used for heterologous expression | was_cloned_into | Xu pXHN ermE-star novN DNA construct | Table 14 reports a 2499-bp AF170880 fragment (15066..17564) from p9-6GE9 inserted into pEM4 BamHI/EcoRI. The table visibly prints BacI-EcoRI for the source fragment, whereas Methods 8.4 says BclI-EcoRI. Preserve the disagreement; a complete experimental junction sequence was not obtained. pEM4 supplies pIJ101 origin, tsr and the ermE* promoter. This is not pXHNEG. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu pXHN ermE-star novN DNA construct | had_reported_transformant_check | pXHN transformant restriction analysis | Figure 13 shows restriction analysis for two transformants, with expected 8.3/2.5-kb EcoRI/PstI fragments and a 10.8-kb SphI band. This is restriction-pattern evidence, not full plasmid sequencing; the subsequent LC-MS experiment is not assigned to a uniquely identified XH12, XH13 or XH14 clone here. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu pXHN ermE-star novN DNA construct | was_introduced_into | S. rishiriensis couN3-mutant production host | The source introduces pXHN into the S. rishiriensis couN3 mutant by protoplast transformation. This heterologous host is separate from the S. lividans protein-purification host and native novobiocin producer. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu pXHN ermE-star novN DNA construct | is_reported_expression_construct_for | pXHN-expressed NovN in the couN3-mutant host | NovN expression is the source intervention. No exact UniProt accession is assigned to this unsequenced expressed product, and the His6 tag of pXHNEG is not transferred to pXHN. Neither expression abundance nor a purified activity measurement is supplied for this host preparation. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| S. rishiriensis couN3-mutant production host | was_used_in | Heterologous NovN coumermycin-analogue experiment | Culture extracts were examined by positive ESI selected-reaction monitoring. The coumermycin A1 comparator uses 1110->282/622; coumermycin D uses 896->175/515; the assigned bis-carbamoylated product uses 982->218/558. These transitions support source assignments, not an independently isolated exact structure. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| pXHN-expressed NovN in the couN3-mutant host | is_intervention_in | Heterologous NovN coumermycin-analogue experiment | The comparison tests heterologous novN expression in the mutant background. It is not direct measurement of native novobiocin biosynthetic flux or a purified turnover constant. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Heterologous NovN coumermycin-analogue experiment | retains_dominant_precursor_signal | Source-assigned coumermycin D precursor signal | Coumermycin D remains the dominant source-assigned metabolite; the carbamoylated species is minor. No absolute pure-product titer or comprehensive conversion percentage is inferred. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Heterologous NovN coumermycin-analogue experiment | has_source_assigned_product_signal | Source-assigned bis-carbamoylated coumermycin D signal | The bis-carbamoylated D signal is reported only in the NovN-expressing mutant. Coumermycin D and its carbamoylated product were not chromatographically separated; SRM distinguishes their assigned ions. Do not represent this as isolation of pure product or two independently measured sequential carbamoylation reactions. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Wild-type and untransformed-mutant comparators | constrain | Heterologous NovN coumermycin-analogue experiment | The assigned carbamoylated signal was not reported for wild type or the untransformed couN3 mutant. These comparator results do not establish the exact intracellular abundance or sequence of NovN. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Heterologous NovN coumermycin-analogue experiment | has_reported_supplementation_test | Nitrogen or carbamoyl-phosphate supplementation readout | Adding 12 mM ammonium nitrate or 4 mM carbamoyl phosphate did not increase the product-to-precursor ratio. The discussion of intracellular cofactor availability is a hypothesis, not a measured limiting pool or demonstrated explanation for low yield. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu pXHN ermE-star novN DNA construct | has_separate_reported_host_test | Reported clorobiocin-mutant negative with data not shown | The source reports no corresponding product from a cloN2 mutant of S. roseochromogenes expressing NovN, explicitly with data not shown. This limited report is not universal NovN nonacceptance of clorobiocin precursors, and the five positive purified-analogue assays remain separate evidence. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Deposited native NovN sequence reference | is_native_reference_for | pXHN-expressed NovN in the couN3-mutant host | UniProtKB:Q9L9F4 is the deposited native sequence reference for pXHN-expressed NovN in the couN3-mutant host (node 'protein'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F4 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 11 edges
Distinct NovN preparation sizes and the explicitly unsolved 2008 crystal forms. Review #560. Xu locators are PDF pages, nineteen higher than printed main-text pages. The owner retains its unspecified sugar stereocentre and SEEDED status; source-assigned novobiocin is not an exact structure-key merge with cathomycin. Native reference, engineered protein and DNA construct are separate entities.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Current deposited native NovN sequence reference | is_gene_product_reference_for | Xu N-terminal His6 NovN preparation | The thesis identifies the protein as novN-derived; current Q9L9F4 is the native reference, not a verified full sequence for the N-terminal His6 sample. Historical sequence-version ambiguity remains. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1 |
| Xu N-terminal His6 NovN preparation | was_measured_by | Xu calibrated NovN size-exclusion result | The methods name Hiload 26/60 Superdex 200 at 1 mL/min and 4 C; Table 19 instead heads the column Sephadex 200. Standards used G1 (50 mM Tris-HCl pH 8, 5 mM DTT, 50 uM PMSF), sample G2 with additional 150 mM NaCl. NovN eluted at 182 mL. Preserve the column-name and sample-versus-standard buffer differences. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Xu calibrated NovN size-exclusion result | supports_source_interpretation | Xu preparation monomer-like hydrodynamic interpretation | The source assigns an apparent 79 kDa, near its 78.5-kDa calculated tagged monomer. Standards were blue dextran 2000 kDa/107.9 mL, aldolase 158/158.6, albumin 66/178.4, ovalbumin 45/212.4 and ribonuclease 13.7/252.4; acetone gave total volume 294 mL. This is preparation-specific hydrodynamic inference, not native in-cell stoichiometry. The reported 79 kDa is not independently recalibrated here: 182 mL lies between the 66- and 45-kDa standard elutions, which were measured in a different buffer from the NovN sample. Preserve that numerical and protocol context rather than silently replacing the reported mass. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| Current deposited native NovN sequence reference | is_native_core_reference_for | 2008 NovN pET37b expression construct | The 2008 source describes novN in pET37b with a C-terminal LEHHHHHHHH extension. Its stated 677-residue native protein plus ten-residue extension gives 687 residues and 75839 Da. No complete experimental vector sequence was independently inspected. | DOI:10.1107/S1744309108030145 |
| 2008 NovN pET37b expression construct | encodes_engineered_protein | 2008 C-terminal LE-His8 NovN preparation | The C-terminal LE-His8 preparation is not the Xu N-terminal His6 fusion. Expression used E. coli BL21(DE3), 1 L LB with 50 mg kanamycin, growth at 310 K to OD600 0.4, 0.1 mM IPTG and overnight expression at 288 K. Nickel affinity and Superdex 200 purification gave about 10 mg/L culture, >95% SDS-PAGE purity and final 10 mg/mL. These are protein yields, not catalytic rates. | DOI:10.1107/S1744309108030145
|
| 2008 C-terminal LE-His8 NovN preparation | was_measured_by | 2008 NovN dynamic-light-scattering result | DLS at 293 K with at least 15 measurements gave 19% polydispersity and apparent mass 160 kDa. These are not the Xu SEC data, and no equilibrium association constant was reported. | DOI:10.1107/S1744309108030145
|
| 2008 NovN dynamic-light-scattering result | supports_source_interpretation | 2008 preparation dimer-like hydrodynamic interpretation | The source compares 160 kDa with the calculated tagged dimer 151.7 kDa. This supports a dimer-like preparation under the reported conditions, not a uniquely established physiological oligomer. | DOI:10.1107/S1744309108030145
|
| Xu preparation monomer-like hydrodynamic interpretation | contributes_to | NovN preparation-specific assembly and tag-attribution limits | The SEC estimate is retained rather than replaced by the later DLS or deposited assembly. Different tags, methods, buffers and sample histories prevent assigning the disagreement to one tested cause. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48601/pdf/Dissertation-endvision.pdf?isAllowed=y&sequence=1
|
| 2008 preparation dimer-like hydrodynamic interpretation | contributes_to | NovN preparation-specific assembly and tag-attribution limits | The later dimer-like estimate does not invalidate the earlier preparation result. Xu PDF p.85 calls the Freel Meyers 2004 preparation N-terminal His8, while the 2008 methods specify C-terminal LE-His8. The complete earlier paper was not inspected; this is an attributed source difference, not a reconstructed 2004 sequence. | DOI:10.1107/S1744309108030145 |
| 2008 C-terminal LE-His8 NovN preparation | formed | 2008 ATP-supplemented unsolved NovN crystal form I | With 1 mM added ATP, form I is reported as C2 with 2.9-A diffraction. Matthews analysis suggests two protomers/asymmetric unit and 52.7% solvent; the self-rotation twofold is 24% of the origin peak. These are crystallographic inferences from an unsolved form, not observed ATP density or an atomic dimer structure. Crystallization methods say 291 K whereas the results say 293 K. | DOI:10.1107/S1744309108030145
|
| 2008 C-terminal LE-His8 NovN preparation | formed | 2008 novobiocin-supplemented unsolved NovN crystal form II | With 1 mM added novobiocin, form II is reported as I222 or I212121, about 2.3-A diffraction (Table 1: 2.29 A), one inferred asymmetric-unit protomer and 62.9% solvent. The paper explicitly plans future phasing. No bound novobiocin, catalytic contacts or identity with the later 9S2T diffraction dataset is asserted. | DOI:10.1107/S1744309108030145
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 10 edges
The 2025 NovN deposit, engineered sequence, coordinate coverage and bounded local geometry. Review #560. Xu locators are PDF pages, nineteen higher than printed main-text pages. The owner retains its unspecified sugar stereocentre and SEEDED status; source-assigned novobiocin is not an exact structure-key merge with cathomycin. Native reference, engineered protein and DNA construct are separate entities.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Current deposited native NovN sequence reference | is_exact_native_core_of | 9S2T deposited C-terminal LE-His8 NovN entity | The 687-residue deposited entity is exactly the 677-aa Q9L9F4 sequence plus LEHHHHHHHH. Its source is S. niveus, taxon 193462, and expression host E. coli BL21(DE3); source strain and vector are unspecified. The native accession is not assigned to the fusion and sequence agreement does not prove it is the exact 2008 sample. | https://files.rcsb.org/download/9S2T.cif |
| 9S2T deposited C-terminal LE-His8 NovN entity | is_deposited_entity_in | 9S2T NovN crystallographic model | 9S2T was released 2025-07-30 with primary citation To Be Published; the 2008 paper is a secondary citation. The I222 model has 2.240-A resolution, Rwork 0.1599 and Rfree 0.2000. This is a public structural deposit, not a claimed new peer-reviewed paper or a retrospective assertion that the 2008 structure was solved. | DOI:10.2210/pdb9S2T/pdb |
| 9S2T NovN crystallographic model | has_coordinate_coverage | 9S2T observed and missing protein coordinates | One asymmetric-unit chain models native residues 1..669 with 5275 protein atom records. Native 670..677 and the ten-residue tag are unmodeled, eighteen residues total; missing tag density is not proof of tag cleavage. Lys444 lacks CD/CE/NZ; Arg601 lacks CG/CD/NE/CZ/NH1/NH2. Eleven protein residues have alternatives: 63,86,116,148,246,315,418,468,501,592,629. | https://files.rcsb.org/download/9S2T.cif
|
| 9S2T NovN crystallographic model | has_author_assigned_assembly | 9S2T author-defined symmetry-generated NovN dimer | Assembly 1 is author-defined with operations x,y,z and -x,-y,z, generating a dimer from one asymmetric-unit chain. Light scattering is the deposited support annotation, not an independently supplied new raw solution dataset or direct native-cell stoichiometry measurement. | https://files.rcsb.org/download/9S2T.cif
|
| 9S2T NovN crystallographic model | contains_deposited_ion | 9S2T partially occupied deposited Fe(III) ion | The single FE residue, author number 706, is modeled at occupancy 0.25. FE(III) is the deposited chemical identity, not an independent determination of oxidation state or evidence that NovN physiologically requires iron. No modeled ATP, Mg or carbamoyl phosphate is present. | https://files.rcsb.org/download/9S2T.cif
|
| 9S2T partially occupied deposited Fe(III) ion | has_selected_coordinate_neighbors | Selected 9S2T Fe-neighbor coordinate distances | Independent Euclidean distances from Fe706 are Asp333 OD1 1.970 A, water842 O 2.190 A, His133 NE2 2.251 A, Asp156 OD1 2.291 A, His137 NE2 2.325 A and water1083 O 2.467 A. The six deposited metalc connections agree. These are coordinate-derived geometry, not metal-binding affinity, catalytic necessity or measured turnover. | https://files.rcsb.org/download/9S2T.cif
|
| 9S2T partially occupied deposited Fe(III) ion | has_validation_context | 9S2T local metal-site validation context | The 2026-03-08 validation report gives Fe706 RSCC 0.98, RSR 0.06 and B 27 A^2 with one atom below 0.9 occupancy, consistent with the CIF 0.25. Its displayed density contours are 0.7 rmsd 2mFo-DFc and 3 rmsd difference density. Asp156 has a CA-CB-CG angle outlier Z 5.44. These quality observations do not independently identify the element, oxidation state or catalytic role. | https://files.rcsb.org/validation/view/9s2t_full_validation.pdf
|
| 9S2T NovN crystallographic model | contains_deposited_ions | Six deposited 9S2T phosphate ions | Six phosphate ions comprise thirty atom records. Validation gives phosphate705 RSCC 0.73, RSR 0.22, B 116-134 A^2, and phosphate707 RSCC 0.80/RSR 0.16. The phosphates are not equally well fitted catalytic groups or evidence for bound carbamoyl phosphate. | https://files.rcsb.org/download/9S2T.cif |
| 9S2T NovN crystallographic model | has_solvent_inventory | 9S2T water-residue and alternate-atom inventory | There are 434 distinct water residues represented by 441 atom records: seven waters have alternate positions. Neither count should be substituted for the other or interpreted as catalytic-water occupancy. | https://files.rcsb.org/download/9S2T.cif |
| 9S2T NovN crystallographic model | constrains_mechanistic_interpretation | Unestablished NovN catalytic-complex and metal requirements | The only modeled nonprotein species are phosphate, Fe and water. No ATP, Mg, carbamoyl phosphate, organic acceptor or novobiocin product is modeled. This deposit therefore does not supply a catalytic substrate complex or establish an iron requirement. The absence of modeled species is not evidence that the enzyme cannot bind them in other conditions. | https://files.rcsb.org/download/9S2T.cif |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 19 nodes, 22 edges
NovH/NovI deposited identities and bounded 2001 activation, loading and hydroxylation evidence. Review #561. The cathomycin/BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status; these precursor assays do not establish an exact-structure merge with fully specified novobiocin. Boll page locators are PDF pages, five above printed pages. Native references, DNA constructs and engineered preparations are distinct.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novH locus | BGC0000834.5 is the owner-linked locus. Current novH is 7934..9736(+), 1803 nt including stop; cluster membership does not determine the owner sugar stereochemistry. | https://mibig.secondarymetabolites.org/go/BGC0000834.5 |
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novI locus | Current novI is 9740..10963(+), 1224 nt including stop, in the same deposited locus. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4
|
| Deposited novH locus | encodes_native_reference | Current deposited native NovH sequence | CDS-aware table-11 translation, including the alternative TTG initiation codon, matches all 600 residues of AAF67501.2 and reviewed Q9L9G0. GenBank identifies S. niveus NCIMB 9219; UniProt uses S. niveus/common S. spheroides, taxon 193462. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novH locus | has_earlier_deposited_translation | Historical deposited NovH sequence | AF170880.2 and .3 encode 600-aa AAF67501.1 at 7882..9684(+). Relative to that version, current NovH has R269P, R271A, R272A and S273V. These are deposit revisions, not independently demonstrated biological alleles. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.2 |
| Deposited novI locus | encodes_native_reference | Deposited native NovI cytochrome P450 sequence | All 407 residues of AAF67502.1 in AF170880.2/.3/.4 match reviewed Q9L9F9 exactly. The earlier CDS coordinates are 9688..10911(+); .4 shifts them by 52 nt without changing this protein. This is native reference identity, not a tagged sample sequence. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Historical deposited NovH sequence | constrains_experimental_identity | Historical strain and experimental sequence limits | Both UniProt entries retain reference strain comments including NCIMB 11891/DSM 40292, whereas the current locus identifies NCIMB 9219. The primary strain correction distinguishes those strains. Neither current sequence nor historical source labels retrospectively sequence the complete 2001 or 2011 preparations. | https://www.uniprot.org/uniprotkb/Q9L9G0 |
| Deposited novH locus | is_reported_source_of | Chen 2001 recombinant NovH preparation | The primary abstract reports E. coli expression, purification and characterization of approximately 65-kDa NovH with predicted A and PCP domains. It does not supply a complete construct or detailed preparation protocol in the inspected text. | DOI:10.1016/S1074-5521(01)00009-6
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| Deposited novI locus | is_reported_source_of | Chen 2001 recombinant NovI preparation | The same abstract reports recombinant approximately 45-kDa NovI as a heme protein functioning as a P450-type monooxygenase. The native accession is not assigned to this incompletely specified preparation. | DOI:10.1016/S1074-5521(01)00009-6
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| Historical strain and experimental sequence limits | limits_accession_assignment_to | Chen 2001 recombinant NovH preparation | Keep the 2001 NovH preparation separate from both native sequence versions and from the later pBB44 His6 preparation. Do not transfer the 2011 host, tag or buffers. | DOI:10.1016/S1074-5521(01)00009-6 |
| Historical strain and experimental sequence limits | limits_accession_assignment_to | Chen 2001 recombinant NovI preparation | Do not infer a particular tag, reductase partner or full construct from the abstract or the matching native NovI reference. | DOI:10.1016/S1074-5521(01)00009-6 |
| Chen 2001 recombinant NovH preparation | catalyzes | Abstract-reported NovH tyrosine adenylation | Purified NovH catalyzes L-tyrosyl-AMP formation through its adenylation domain. No detailed kinetic condition or negative substrate panel is extracted from the abstract. | DOI:10.1016/S1074-5521(01)00009-6
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| L-Tyrosine supplied for NovH activation | is_substrate_for | Abstract-reported NovH tyrosine adenylation | L-Tyrosine is the named activation substrate, not free beta-hydroxytyrosine. | DOI:10.1016/S1074-5521(01)00009-6
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| Abstract-reported NovH tyrosine adenylation | forms | L-Tyrosyl-AMP intermediate | The reported activated intermediate is L-tyrosyl-AMP. This precursor activation does not establish mature antibiotic formation. | DOI:10.1016/S1074-5521(01)00009-6
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| Chen 2001 recombinant NovH preparation | can_be_phosphopantetheinylated_to | Phosphopantetheinylated NovH PCP state | The abstract reports posttranslational phosphopantetheinylation on the PCP domain. The modifying enzyme and conditions are not supplied here; the later Sfp coexpression protocol is not silently substituted. | DOI:10.1016/S1074-5521(01)00009-6
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| Phosphopantetheinylated NovH PCP state | accumulates_covalently_loaded_state | Covalently loaded L-tyrosyl-S-NovH | The reported L-tyrosyl-S-enzyme intermediate is covalently attached to the holo PCP domain. It is not a free tyrosine product or an assembly stoichiometry measurement. | DOI:10.1016/S1074-5521(01)00009-6
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| L-Tyrosyl-AMP intermediate | is_activation_intermediate_for | Covalently loaded L-tyrosyl-S-NovH | The abstract connects NovH adenylation with accumulation of the covalently loaded holo-PCP intermediate; no additional rate or independently resolved coupling stoichiometry is asserted. | DOI:10.1016/S1074-5521(01)00009-6
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| Chen 2001 recombinant NovI preparation | catalyzes | NovI-catalyzed tethered-tyrosine hydroxylation | NovI acts as the reported P450 monooxygenase on the NovH-tethered tyrosyl acyl enzyme. | DOI:10.1016/S1074-5521(01)00009-6
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| Covalently loaded L-tyrosyl-S-NovH | is_substrate_for | NovI-catalyzed tethered-tyrosine hydroxylation | The abstract specifies tyrosyl-S-NovH as the substrate. This statement is not expanded into an uninspected quantitative free-amino-acid rejection panel. | DOI:10.1016/S1074-5521(01)00009-6
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| NovI-catalyzed tethered-tyrosine hydroxylation | forms | Beta-hydroxytyrosyl-S-NovH | The reported product remains attached to NovH as beta-hydroxytyrosyl-S-NovH. | DOI:10.1016/S1074-5521(01)00009-6
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| Beta-hydroxytyrosyl-S-NovH | was_examined_by | Alkaline release and product analysis | Alkaline release, HPLC analysis of radioactive [3H]beta-hydroxytyrosine and mass spectrometry support the abstract-reported product assignment. | DOI:10.1016/S1074-5521(01)00009-6
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| Alkaline release and product analysis | supports_stereochemical_assignment_of | Released (3R,2S)-3-hydroxytyrosine | Comparison with authentic standards assigns the released amino acid as (3R,2S)-3-hydroxytyrosine. This assignment concerns the released precursor, not the owner sugar stereocentre. | DOI:10.1016/S1074-5521(01)00009-6
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| Alkaline release and product analysis | also_detected | Detected 4-hydroxybenzaldehyde breakdown product | The abstract reports 4-hydroxybenzaldehyde as a retro-aldol breakdown product of beta-hydroxytyrosine, not a second direct NovI-catalyzed product. | DOI:10.1016/S1074-5521(01)00009-6
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 13 nodes, 14 edges
NovH sequence-version limits, literal primers and the two 2011 host-conditioned preparations. Review #561. The cathomycin/BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status; these precursor assays do not establish an exact-structure merge with fully specified novobiocin. Boll page locators are PDF pages, five above printed pages. Native references, DNA constructs and engineered preparations are distinct.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Current deposited native NovH reference | has_historical_reference_version | Historical NovH sequence named by the 2011 study | Current 600-aa Q9L9G0/AAF67501.2 differs at four residues from the AAF67501.1 version. Both are sequence references, not identities for the His6 fusion. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Historical NovH sequence named by the 2011 study | matches_selected_window_in | Selected printed NovH alignment window | Figure S1 NovH residues 261..280 read GRVSHAVLRPRRSATMSPDA, matching the historical sequence rather than current GRVSHAVLPPAAVATMSPDA. Met383 is retained in both. This is a checked 20-residue window, not a claim to have transcribed the entire 600-aa printed alignment or sequenced pBB44. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| Historical NovH sequence named by the 2011 study | is_sequence_reference_for | pBB44 pET28a NovH expression construct | The study names NovH accession AAF67501, amplifies novH from a cluster-containing cosmid and clones via NdeI/XhoI into pET28a as pBB44. Entire-gene sequencing is reported, but its complete experimental plasmid sequence was not independently obtained. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| pBB44 pET28a NovH expression construct | has_checked_primer_model | Literal 2011 NovH primer model | Literal forward GGGAATTCCATATGTTCAACACACGTGCGAAC and reverse GCCCTCGAGTCACTCCTCCAGGGTCGCTA give a modeled 1823-nt PCR product including 11-nt and 9-nt tails around the 1803-nt CDS. They recode the native TTG start to ATG and CDS nucleotide 1794 from C to G (Leu598 CTC to CTG). Table-11 translation preserves each version-specific 600-aa core; no missense mutation, complete vector-derived tag or scar is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| pBB44 pET28a NovH expression construct | encodes_engineered_preparation | pBB44 His6-NovH preparation from deletion host | The pET28a construct supplies N-terminal His6-NovH. Unlike the MbtH partners, the adenylating enzymes retain their tags. This sample is not native Q9L9G0. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| pBB44 pET28a NovH expression construct | encodes_engineered_preparation | pBB44 His6-NovH preparation from intact-ybdZ host | The same reported construct was expressed in the intact-ybdZ host. Shared construct design does not make this preparation compositionally identical to protein purified from the deletion host. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| BL21(DE3) ybdZ-deletion expression host | expressed | pBB44 His6-NovH preparation from deletion host | The ybdZ::acc(3)IV host was made by Red/ET recombination with an apramycin cassette and PCR-confirmed genotype. Expression used 35 mL LB inoculum into 1 L TB, 50 ug/mL kanamycin and 25 ug/mL chloramphenicol, growth at 37 C to OD600 0.6, then 0.4 mM IPTG for 14 h at 20 C. These are protein-production conditions, not a native antibiotic-production intervention. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| BL21(DE3) intact-ybdZ expression host | expressed | pBB44 His6-NovH preparation from intact-ybdZ host | NovH was also purified from the unmodified BL21(DE3) host using the reported expression workflow; endogenous YbdZ availability is a preparation variable, not evidence of its stoichiometric presence in the eluate. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| B. subtilis Sfp supplied by pSU20_sfp | was_coexpressed_for_holoenzyme_preparation | pBB44 His6-NovH preparation from deletion host | Both host backgrounds carry pSU20_sfp encoding B. subtilis Sfp phosphopantetheinyl transferase. The stated purpose is holoenzyme expression; no site-occupancy percentage or exact assayed Sfp accession is supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| B. subtilis Sfp supplied by pSU20_sfp | was_coexpressed_for_holoenzyme_preparation | pBB44 His6-NovH preparation from intact-ybdZ host | Sfp coexpression also applies to the intact-ybdZ preparation. It is not an endogenous novobiocin-cluster protein or a measured mature-product assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| pBB44 His6-NovH preparation from deletion host | was_isolated_by | Ni-NTA and preparative Superdex purification | Ni-NTA purification was followed by HiLoad 26/60 Superdex 200 in 20 mM Tris-HCl pH 8, 150 mM NaCl and 2 mM DTT. Protein was concentrated with a 10,000-MWCO filter and stored at -80 C; the His6 tag was retained. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| pBB44 His6-NovH preparation from intact-ybdZ host | was_isolated_by | Ni-NTA and preparative Superdex purification | The same stated purification workflow applies to the intact-host NovH preparation; it does not independently exclude copurifying proteins. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| Ni-NTA and preparative Superdex purification | gave_reported_yields | NovH preparation yields and concentration method | NovH yield was 38.6 mg/L culture from the deletion host and 64 mg/L from the intact-ybdZ host. Concentration was estimated at A280 using calculated extinction coefficients. These are not active-protein fractions, enzyme specific activities or natural-product titers. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| pBB44 His6-NovH preparation from deletion host | has_displayed_gel_readout | Figure S2 NovH SDS-PAGE readout | Figure S2 shows NovH purified from the deletion host, stained with Coomassie Brilliant Blue R-250; its caption gives 65.7 kDa as the calculated tagged mass. A gel band does not establish a native oligomer or a measured purity percentage. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 15 edges
Three preparation-conditioned NovH kinetic fits, inferred YbdZ carryover and bounded homology models. Review #561. The cathomycin/BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status; these precursor assays do not establish an exact-structure merge with fully specified novobiocin. Boll page locators are PDF pages, five above printed pages. Native references, DNA constructs and engineered preparations are distinct.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| 2011 His6-NovH from deletion host | was_assayed_by | ATP-[32P]PPi exchange assay | The default 100-uL exchange assay contains 95 mM Tris-HCl pH 8, 5 mM MgCl2, 5 mM TCEP, 2 mM ATP, 1.5 mM L-tyrosine, 1 uM adenylating enzyme and 1 mM [32P]pyrophosphate, with 1.2 uM MbtH partner when included. Incubation is 5 min at 30 C; charcoal capture and scintillation measure exchanged label. This is not a direct PCP-loading or mature-product flux assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| 2011 His6-NovH from intact-ybdZ host | was_assayed_by | ATP-[32P]PPi exchange assay | The intact-host preparation was compared by the same exchange method, not a separate native producer experiment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| 2011 processed CloY preparation | was_added_in | ATP-[32P]PPi exchange assay | The NovH/CloY kinetic comparison used the reported 1:1.2 enzyme:partner molar ratio. Addition does not itself measure binding affinity or complex stoichiometry. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| ATP-[32P]PPi exchange assay | gave_deletion_host_fit | NovH-alone deletion-host kinetic fit | NovH alone from the deletion host has Figure S3 Km 274.5 +/- 39.5 uM for L-tyrosine, kcat 0.079 s^-1 and kcat/Km 290 s^-1 M^-1. Table 1 rounds Km to 275 uM. Nonlinear fits used Prism 5.0 and the method reports means of two independent reactions. The displayed plus/minus statistic is not defined in the inspected methods/caption; do not relabel it SD or SEM. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| 2011 His6-NovH from deletion host | retains_activity_in | NovH-alone deletion-host kinetic fit | Deletion-host NovH is active without added MbtH protein, as also shown in Figure 3. This is a positive result in this preparation, not proof that every NovH preparation or native-cell pathway is MbtH-independent. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| ATP-[32P]PPi exchange assay | gave_intact_host_fit | NovH-alone intact-host kinetic fit | NovH alone from intact-ybdZ E. coli has Km 1278 +/- 106 uM, kcat 0.120 s^-1 and kcat/Km 94 s^-1 M^-1. Figure S3 prints kcat as 0.12. The same uncertainty-definition limitation applies. These values are not assigned to the deletion-host protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| ATP-[32P]PPi exchange assay | gave_cloy_supplemented_fit | NovH plus CloY kinetic fit | Deletion-host NovH plus CloY has Figure S3 Km 276.5 +/- 20.5 uM, kcat 0.438 s^-1 and kcat/Km 1580 s^-1 M^-1; Table 1 rounds Km to 277 uM. The close Km values and increased turnover are source-reported assay comparisons, not a direct observation of a catalytic conformational change. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| NovH-alone intact-host kinetic fit | motivates_source_hypothesis | Inferred YbdZ explanation for host-dependent kinetics | The authors infer YbdZ copurification from the host-dependent NovH kinetics. They compare the intact-host Km with the 1390-uM value cited from Chen 2001; that historical value is not independently re-extracted from the unobtained full 2001 methods. Higher kcat coincides with lower reported catalytic efficiency because Km also rises. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| Endogenous E. coli YbdZ candidate copurifying partner | is_proposed_copurifying_factor_in | Inferred YbdZ explanation for host-dependent kinetics | YbdZ is the named endogenous host MbtH-like protein. The NovH comparison does not directly identify or quantify a copurifying YbdZ band or supply a NovH-YbdZ binding constant. No convenient K-12 accession is substituted for the incompletely sequenced BL21(DE3) experimental preparation. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| NovH-alone deletion-host kinetic fit | motivated_computational_comparison | PheA-based NovH/CloH homology models | The 2011 authors modeled NovH and CloH after the PheA adenylation domain of GrsA, naming PDB 1AMU. Their statement that no experimental NovH/CloH structure was available is historical, not a current exhaustive structure search. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| PheA-based NovH/CloH homology models | supports_source_hypothesis | Proposed Met383-linked active-site conformation | The model compares NovH Met383, CloH Leu383 and PheA Lys396, proposing effects on Tyr397/Glu316 positioning near the Mg site. These are modeled positions, not experimentally observed NovH contacts. The reported L383M intervention was in CloH, not a NovH mutant. No CloH mutant activity is reassigned to a NovH sequence or to this compound owner. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| Inferred YbdZ explanation for host-dependent kinetics | does_not_resolve | Unresolved NovH partner binding and complex stoichiometry | Activity stimulation and inferred copurification do not determine NovH oligomer stoichiometry. The analytical SEC mass and proposed 2:2 complex in this study concern SimH/SimY; the CloH/CloY coexpression experiments also remain separate. Neither is transferred as a measured NovH assembly. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| Deposited native NovH sequence reference | is_native_reference_for | 2011 His6-NovH from deletion host | UniProtKB:Q9L9G0 is the deposited native sequence reference for 2011 His6-NovH from deletion host (node 'delta'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9G0 |
| Deposited native NovH sequence reference | is_native_reference_for | 2011 His6-NovH from intact-ybdZ host | UniProtKB:Q9L9G0 is the deposited native sequence reference for 2011 His6-NovH from intact-ybdZ host (node 'intact'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9G0 |
| Deposited native CloY sequence reference | is_native_reference_for | 2011 processed CloY preparation | UniProtKB:Q8GHC8 is the deposited native sequence reference for 2011 processed CloY preparation (node 'cloy'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q8GHC8 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 17 nodes, 20 edges
Four heterologous MbtH partners, exact native reference boundaries and complete NovH cross-activation scope. Review #561. The cathomycin/BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status; these precursor assays do not establish an exact-structure merge with fully specified novobiocin. Boll page locators are PDF pages, five above printed pages. Native references, DNA constructs and engineered preparations are distinct.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| 2011 deletion-host His6-NovH preparation | was_tested_in | Four-partner NovH cross-activation assay | Figure 6 tests deletion-host NovH with CloY, CdaX, SimY or Orf1van at enzyme:partner 1:1.2 by the 5-min, 30-C ATP-PPi exchange assay. The panel is heterologous functional stimulation, not four endogenous novobiocin-cluster partners or direct physical binding measurements. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Deposited CloY sequence reference | is_native_reference_for | pBB37-derived processed CloY preparation | CloY is 71 aa: AF329398.1 positions 12920..13135(+), AAN65223.1 and Q8GHC8 match exactly. The GenBank source is S. roseochromogenus subsp. oscitans DS 12.976 (strain taxon 1352936); UniProt uses subspecies taxon 149682. The paper amplifies cloY from a cluster-containing cosmid for pBB37. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| Deposited SimY sequence reference | is_native_reference_for | pBB28-derived SimY preparation with tag-stage limitation | SimY Q9F5J2 exactly matches the 70-aa AAL15602.1 translation in AF322256.1 and the previously checked AAG34186.1/AAK06806.1 deposits. The paper names AAG34186 and uses a sim-cluster cosmid for pBB28; the native accession does not denote the processed fusion. | https://www.ncbi.nlm.nih.gov/nuccore/AF322256.1 |
| Deposited CdaX SCO3218 sequence reference | is_native_reference_for | pBB32-derived processed CdaX preparation | CdaX is the source-named CAB38589.1/SCO3218, 71 aa, exactly matching Q9Z388 and CDS-aware translation of AL939115.1:183050..183265(-). The genomic GTG start translates as initiator methionine. UniProt names S. coelicolor A3(2)/M145, taxon 100226, whereas the paper amplified cdaX from M512 to make pBB32. The reference match is not independent sequencing of the complete M512-derived experimental construct. | https://www.ncbi.nlm.nih.gov/protein/CAB38589.1 |
| Deposited Orf1van sequence reference | is_native_reference_for | pBB34-derived processed synthetic Orf1van preparation | The source-named AAL90876.1 is a 69-aa conceptual translation from Amycolatopsis orientalis, taxon 31958, at AF486630.1:720..929(+). The 210-nt CDS including stop matches the protein deposit. The paper used a commercially synthesized, E. coli-codon-optimized orf1van in pET28a/pBB34, not a directly assayed native preparation. No full synthetic construct sequence or defensible exact UniProt mapping was obtained in the bounded accession queries; the named NCBI accession is retained. | https://www.ncbi.nlm.nih.gov/protein/AAL90876.1 |
| Deposited SimY sequence reference | exposes_reported_length_difference | SimY and Orf1van reported-length discrepancies | The paper lists SimY as 69 aa and Orf1van as 70 aa, whereas the named native deposits give SimY 70 aa and Orf1van 69 aa. No initiator-removal, swapped-label correction or altered experimental sequence is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| Deposited Orf1van sequence reference | also_exposes_reported_length_difference | SimY and Orf1van reported-length discrepancies | The Orf1van discrepancy concerns the native accession versus the source description; it does not establish the length of an independently sequenced thrombin-processed synthetic protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| MbtH partner expression and thrombin-processing workflow | has_checked_primer_models | Literal CloY, SimY and CdaX primer models | The six printed CloY/SimY/CdaX primers were visually checked and modeled against their native CDSs. PCR lengths including 11-nt forward and 9-nt reverse tails are 236, 233 and 236 nt, respectively. CloY and SimY cores are unchanged; CdaX GTG is recoded to ATG without changing its CDS-aware protein translation. These models do not establish complete vector junctions or post-thrombin scars. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| MbtH partner expression and thrombin-processing workflow | produced | pBB37-derived processed CloY preparation | This partner was expressed in E. coli Rosetta2(DE3) as an N-terminal His6 fusion. Methods report thrombin removal of MbtH tags (0.4 U/mg, 8 h at 4 C) before gel filtration, with Ni-NTA and Superdex purification. The reported preparation yield is 6.9 mg/L culture. A280-derived concentration and tag removal do not establish active fraction or restoration of the exact native N terminus. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
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| pBB37-derived processed CloY preparation | stimulated_novh_in | Four-partner NovH cross-activation assay | Figure 6 shows higher NovH exchange activity with this partner than without added MbtH. The NovH panel normalizes its CloY condition to 100%; the other enzyme panels have their own denominators. No precise percentage is newly estimated from the bars, and stimulation alone is not direct binding or native pathway flux. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| MbtH partner expression and thrombin-processing workflow | produced | pBB28-derived SimY preparation with tag-stage limitation | This partner was expressed in E. coli Rosetta2(DE3) as an N-terminal His6 fusion. Methods report thrombin removal of MbtH tags (0.4 U/mg, 8 h at 4 C) before gel filtration, with Ni-NTA and Superdex purification. The reported preparation yield is 27 mg/L culture. A280-derived concentration and tag removal do not establish active fraction or restoration of the exact native N terminus. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB28-derived SimY preparation with tag-stage limitation | stimulated_novh_in | Four-partner NovH cross-activation assay | Figure 6 shows higher NovH exchange activity with this partner than without added MbtH. The NovH panel normalizes its CloY condition to 100%; the other enzyme panels have their own denominators. No precise percentage is newly estimated from the bars, and stimulation alone is not direct binding or native pathway flux. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| MbtH partner expression and thrombin-processing workflow | produced | pBB32-derived processed CdaX preparation | This partner was expressed in E. coli Rosetta2(DE3) as an N-terminal His6 fusion. Methods report thrombin removal of MbtH tags (0.4 U/mg, 8 h at 4 C) before gel filtration, with Ni-NTA and Superdex purification. The reported preparation yield is 23.8 mg/L culture. A280-derived concentration and tag removal do not establish active fraction or restoration of the exact native N terminus. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB32-derived processed CdaX preparation | stimulated_novh_in | Four-partner NovH cross-activation assay | Figure 6 shows higher NovH exchange activity with this partner than without added MbtH. The NovH panel normalizes its CloY condition to 100%; the other enzyme panels have their own denominators. No precise percentage is newly estimated from the bars, and stimulation alone is not direct binding or native pathway flux. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| MbtH partner expression and thrombin-processing workflow | produced | pBB34-derived processed synthetic Orf1van preparation | This partner was expressed in E. coli Rosetta2(DE3) as an N-terminal His6 fusion. Methods report thrombin removal of MbtH tags (0.4 U/mg, 8 h at 4 C) before gel filtration, with Ni-NTA and Superdex purification. The reported preparation yield is 30.2 mg/L culture. A280-derived concentration and tag removal do not establish active fraction or restoration of the exact native N terminus. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB34-derived processed synthetic Orf1van preparation | stimulated_novh_in | Four-partner NovH cross-activation assay | Figure 6 shows higher NovH exchange activity with this partner than without added MbtH. The NovH panel normalizes its CloY condition to 100%; the other enzyme panels have their own denominators. No precise percentage is newly estimated from the bars, and stimulation alone is not direct binding or native pathway flux. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB28-derived SimY preparation with tag-stage limitation | has_tag_stage_ambiguity | Figure S2 SimY tag-stage ambiguity | Methods report cleavage of MbtH tags, but Figure S2 explicitly labels N-His6-SimY and gives 9.4 kDa. These may represent different preparation stages; the figure does not independently prove that the activation assays used retained-tag SimY or that cleavage restored an exact native sequence. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| MbtH-only assay control | constrains_interpretation_of | Four-partner NovH cross-activation assay | The results report no activity from MbtH-like proteins alone. This is the study-level control for exchange activity, not a separately quantified zero with a supplied detection limit for every Figure 6 pair. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Four-partner NovH cross-activation assay | supports_limited_partner_scope | Bounded heterologous activation without native-partner assignment | All four partners stimulated NovH and CloY gave the highest displayed activity. The 2011 partial-genome discussion reports no mbtH-like gene in the nov cluster/immediate vicinity but at least two distant orthologs. That historical, strain-label-qualified observation is not a present-day genome-wide absence claim or assignment of the tested heterologous partners as native NovH cofactors. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| Deposited Orf1van sequence reference | has a sequence-identical UniProtKB example | Orf1van (Amycolatopsis orientalis UniProtKB example) | UniProtKB:Q8RN06 (Amycolatopsis orientalis Protein mbtH) shares 100% amino-acid sequence identity (69 aa, UPI00000B5FEE) with AF486630.1 AAL90876.1 (Orf1van). | https://www.uniprot.org/uniprotkb/Q8RN06 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 18 nodes, 19 edges
Exact native NovJ/NovK identities and bounded primary evidence for tethered precursor oxidation. Review #562. The cathomycin/BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status. Precursor and recombinant protein evidence does not resolve that stereocentre or establish an exact-key merge with fully specified novobiocin. Main-paper evidence is abstract-only; the official four-page supplement and native deposits were inspected directly.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novJ locus | BGC0000834.5 links this owner to AF170880.4. The novJ CDS is 10983..11771(+), 789 nt including stop. | https://mibig.secondarymetabolites.org/go/BGC0000834.5 |
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novK locus | The novK CDS is 11768..12502(+), 735 nt including stop, in the same deposit. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4
|
| Deposited novJ locus | encodes_native_reference | Deposited native NovJ sequence reference | All 262 residues of AAF67503.1 match Q9L9F8 and CDS-aware table-11 translations of AF170880.2/.3/.4. Earlier coordinates are 10931..11719(+). This establishes deposited native sequence identity, not an engineered sample sequence. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novK locus | encodes_native_reference | Deposited native NovK sequence reference | All 244 residues of AAF67504.1 match Q9L9F7 and all three native CDS translations. Earlier coordinates are 11716..12450(+). No tag or engineered sequence is included in this native accession. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited native NovJ sequence reference | has_provenance_limit | Unresolved historical versus experimental strain labels | Current AF170880.4 identifies S. niveus NCIMB 9219. UniProt uses S. niveus/common S. spheroides, taxon 193462, but retains historical reference strain comments including ATCC 23965 and NCIMB 11891. SI p.3 instead prints ATCC26935. No synonymy, typographical repair or uniquely identified experimental isolate is inferred. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited native NovK sequence reference | shares_provenance_limit | Unresolved historical versus experimental strain labels | NovK has the same current-versus-historical metadata boundary. The matching native sequences do not resolve which complete engineered preparation was used for each published experiment. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novJ locus | is_named_source_of | Recombinant NovJ constituent of the reported complex | The abstract identifies recombinant NovJ in the purified joint preparation; it does not establish separate soluble purification, tag, full fusion sequence or exact isolate for this constituent. | DOI:10.1021/bi051297m
|
| Deposited novK locus | is_named_source_of | Recombinant NovK constituent of the reported complex | Recombinant NovK is the other named constituent. Individual-construct SI tag labels are not transferred to the main preparation. | DOI:10.1021/bi051297m
|
| E. coli host reported for the NovJ/NovK preparation | was_expression_host_for | Abstract-reported recombinant NovJ2K2 heterotetramer | The joint preparation was expressed in and purified from E. coli. The inspected abstract does not supply host strain, yield or purity. | DOI:10.1021/bi051297m
|
| Recombinant NovJ constituent of the reported complex | is_reported_constituent_of | Abstract-reported recombinant NovJ2K2 heterotetramer | The paper reports J2K2, hence two NovJ subunits in its assigned assembly. This is source-reported stoichiometry, not an independent mass or interface determination. | DOI:10.1021/bi051297m
|
| Recombinant NovK constituent of the reported complex | is_reported_constituent_of | Abstract-reported recombinant NovJ2K2 heterotetramer | The same reported assembly contains two NovK subunits. The native UniProt accession is not an identifier for the whole complex. | DOI:10.1021/bi051297m
|
| Abstract-reported recombinant NovJ2K2 heterotetramer | catalyzes | Benzylic oxidation of NovH-tethered beta-hydroxytyrosine | The reaction oxidizes the tethered tyrosyl beta-alcohol. It is not demonstration of free-tyrosine oxidation, spontaneous ring closure or mature natural-product synthesis. | DOI:10.1021/bi051297m
|
| Beta-hydroxytyrosyl-S-NovH substrate | is_substrate_for | Benzylic oxidation of NovH-tethered beta-hydroxytyrosine | The substrate remains a phosphopantetheinyl thioester on NovH. No exact NovH tag, sequence version or preparation is inferred here. | DOI:10.1021/bi051297m
|
| NADP electron acceptor | is_electron_acceptor_for | Benzylic oxidation of NovH-tethered beta-hydroxytyrosine | The primary source specifies oxidized NADP as electron acceptor, not NADPH as reducing cosubstrate. Concentrations and cofactor specificity panels were not obtained. | DOI:10.1021/bi051297m
|
| Benzylic oxidation of NovH-tethered beta-hydroxytyrosine | forms | Beta-ketotyrosyl-S-NovH product | The assigned product is beta-ketotyrosyl-S-NovH; no separate experimentally established cyclase is added from this abstract. | DOI:10.1021/bi051297m
|
| Beta-ketotyrosyl-S-NovH product | was_subject_to | Hydrolytic release from the NovH carrier | The source reports hydrolysis to analyze the carrier-bound moiety. Hydrolysis conditions and recovery yield remain uninspected. | DOI:10.1021/bi051297m
|
| Hydrolytic release from the NovH carrier | released | Released labile beta-ketotyrosine analyte | The analyte is labile beta-ketotyrosine, not an independently grounded mature aminocoumarin structure. | DOI:10.1021/bi051297m
|
| Released labile beta-ketotyrosine analyte | was_identified_by | Mass analysis and synthetic-comparator identification | Mass spectroscopy and a synthetic comparator support the product assignment. No uninspected m/z, chromatographic trace or uncertainty is newly asserted. | DOI:10.1021/bi051297m
|
| Conflicting UniProt reaction-direction wording | must_not_reverse | Benzylic oxidation of NovH-tethered beta-hydroxytyrosine | Both inspected UniProt function paragraphs describe NADPH-dependent reduction while naming a beta-keto product. The directly inspected primary abstract instead reports NADP-dependent oxidation. Preserve the discrepancy; do not create a second demonstrated reverse reaction. | https://www.uniprot.org/uniprotkb/Q9L9F8 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 15 nodes, 20 edges
Four reported individual NovJ/NovK constructs, intended tagged products and bounded primer/solubility evidence. Review #562. The cathomycin/BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status. Precursor and recombinant protein evidence does not resolve that stereocentre or establish an exact-key merge with fully specified novobiocin. Main-paper evidence is abstract-only; the official four-page supplement and native deposits were inspected directly.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| SI-labeled S. spheroides ATCC26935 DNA source | was_reported_dna_source_for | pNovJ-N-pET16b DNA construct | The SI reports genomic PCR with Pfu Turbo for pNovJ-N-pET16b. ATCC26935 is retained literally, without silently changing it to ATCC 23965 or NCIMB 9219. | DOI:10.1021/bi051297m.s001
|
| pNovJ-N-pET16b DNA construct | specifies_intended_tagged_product | Intended N-terminal His10 NovJ product | The reported N-terminal His10 architecture is a construct description, not a fully sequenced fusion or proof of successful soluble protein recovery. No native UniProt accession is assigned to it. | DOI:10.1021/bi051297m.s001
|
| pNovJ-N-pET16b DNA construct | was_screened_in | Individual-construct solubility screening | This is one of the four individually expressed constructs named in the small-scale solubility screen, not necessarily the construct used for the reported joint complex. | DOI:10.1021/bi051297m.s001
|
| Deposited native NovJ sequence reference | is_sequence_reference_for | pNovJ-N-pET16b DNA construct | The literal novJ-1/novJ-2 model against AF170880.2/.3/.4 gives an 816-nt PCR product including a 12-nt forward tail, preserves the stop codon, and preserves the 262-aa native core. No corrected restriction site, complete vector junction or experimental fusion sequence is invented. | DOI:10.1021/bi051297m.s001 |
| NovJ primer versus restriction-method discrepancy | limits_reconstruction_of | pNovJ-N-pET16b DNA construct | The printed NovJ reverse primer contains TTCGAA (BstBI), not AAGCTT (HindIII). Its complete literal PCR model lacks HindIII although the prose reports NdeI/HindIII digestion. This is a source inconsistency, not evidence that the reported construct never existed. | DOI:10.1021/bi051297m.s001 |
| SI-labeled S. spheroides ATCC26935 DNA source | was_reported_dna_source_for | pNovJ-C-pET37b DNA construct | The SI reports genomic PCR with Pfu Turbo for pNovJ-C-pET37b. ATCC26935 is retained literally, without silently changing it to ATCC 23965 or NCIMB 9219. | DOI:10.1021/bi051297m.s001
|
| pNovJ-C-pET37b DNA construct | specifies_intended_tagged_product | Intended C-terminal His8 NovJ product | The reported C-terminal His8 architecture is a construct description, not a fully sequenced fusion or proof of successful soluble protein recovery. No native UniProt accession is assigned to it. | DOI:10.1021/bi051297m.s001
|
| pNovJ-C-pET37b DNA construct | was_screened_in | Individual-construct solubility screening | This is one of the four individually expressed constructs named in the small-scale solubility screen, not necessarily the construct used for the reported joint complex. | DOI:10.1021/bi051297m.s001
|
| Deposited native NovJ sequence reference | is_sequence_reference_for | pNovJ-C-pET37b DNA construct | The literal novJ-1/novJ-3 model against AF170880.2/.3/.4 gives an 816-nt PCR product including a 12-nt forward tail, omits the native stop codon, and preserves the 262-aa native core. No corrected restriction site, complete vector junction or experimental fusion sequence is invented. | DOI:10.1021/bi051297m.s001 |
| NovJ primer versus restriction-method discrepancy | limits_reconstruction_of | pNovJ-C-pET37b DNA construct | The printed NovJ reverse primer contains TTCGAA (BstBI), not AAGCTT (HindIII). Its complete literal PCR model lacks HindIII although the prose reports NdeI/HindIII digestion. This is a source inconsistency, not evidence that the reported construct never existed. | DOI:10.1021/bi051297m.s001 |
| SI-labeled S. spheroides ATCC26935 DNA source | was_reported_dna_source_for | pNovK-N-pET16b DNA construct | The SI reports genomic PCR with Pfu Turbo for pNovK-N-pET16b. ATCC26935 is retained literally, without silently changing it to ATCC 23965 or NCIMB 9219. | DOI:10.1021/bi051297m.s001
|
| pNovK-N-pET16b DNA construct | specifies_intended_tagged_product | Intended N-terminal His10 NovK product | The reported N-terminal His10 architecture is a construct description, not a fully sequenced fusion or proof of successful soluble protein recovery. No native UniProt accession is assigned to it. | DOI:10.1021/bi051297m.s001
|
| pNovK-N-pET16b DNA construct | was_screened_in | Individual-construct solubility screening | This is one of the four individually expressed constructs named in the small-scale solubility screen, not necessarily the construct used for the reported joint complex. | DOI:10.1021/bi051297m.s001
|
| Deposited native NovK sequence reference | is_sequence_reference_for | pNovK-N-pET16b DNA construct | The literal novK-1/novK-2 model gives 759 nt including the 15-nt forward tail and retains the native stop. Both reverse primers change CDS nucleotide 726 C to T, recoding Arg242 CGC to CGT without changing the 244-aa protein core. NdeI/HindIII sites are present; a complete vector fusion or processed terminus is not established. | DOI:10.1021/bi051297m.s001 |
| SI-labeled S. spheroides ATCC26935 DNA source | was_reported_dna_source_for | pNovK-C-pET37b DNA construct | The SI reports genomic PCR with Pfu Turbo for pNovK-C-pET37b. ATCC26935 is retained literally, without silently changing it to ATCC 23965 or NCIMB 9219. | DOI:10.1021/bi051297m.s001
|
| pNovK-C-pET37b DNA construct | specifies_intended_tagged_product | Intended C-terminal His8 NovK product | The reported C-terminal His8 architecture is a construct description, not a fully sequenced fusion or proof of successful soluble protein recovery. No native UniProt accession is assigned to it. | DOI:10.1021/bi051297m.s001
|
| pNovK-C-pET37b DNA construct | was_screened_in | Individual-construct solubility screening | This is one of the four individually expressed constructs named in the small-scale solubility screen, not necessarily the construct used for the reported joint complex. | DOI:10.1021/bi051297m.s001
|
| Deposited native NovK sequence reference | is_sequence_reference_for | pNovK-C-pET37b DNA construct | The literal novK-1/novK-3 model gives 756 nt including the 15-nt forward tail and omits the native stop. Both reverse primers change CDS nucleotide 726 C to T, recoding Arg242 CGC to CGT without changing the 244-aa protein core. NdeI/HindIII sites are present; a complete vector fusion or processed terminus is not established. | DOI:10.1021/bi051297m.s001 |
| E. coli BL21(DE3) expression-screen host | was_used_for | Individual-construct solubility screening | The SI reports BL21(DE3), LB with 50 ug/mL kanamycin for pET37b and pET28b or 100 ug/mL ampicillin for pET16b. The four named individual constructs were screened at 37, 30, 25 and 16 C with and without IPTG. Mention of pET28b does not supply an additional fully described individual construct here. | DOI:10.1021/bi051297m.s001
|
| Individual-construct solubility screening | does_not_establish | Unresolved fusion sequence and positive-expression outcomes | The stated aim is to find soluble-expression conditions; the inspected SI does not report a successful condition, yield, purity or activity for each individual construct. Induction dose/time and complete plasmid sequences also remain uninspected. A screening design is not a positive protein-production measurement. | DOI:10.1021/bi051297m.s001
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 9 nodes, 10 edges
Source-reported J2K2 assembly and three directly inspected concentration-labeled SEC traces. Review #562. The cathomycin/BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status. Precursor and recombinant protein evidence does not resolve that stereocentre or establish an exact-key merge with fully specified novobiocin. Main-paper evidence is abstract-only; the official four-page supplement and native deposits were inspected directly.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| NovJ/NovK sample used for supporting gel-filtration traces | was_analyzed_by | Analytical gel filtration of NovJ/NovK samples | Supporting Figures 1-3 plot analytical gel-filtration absorbance against retention volume. The four SI pages do not identify a complete sample sequence, unique tag combination, buffer, column or mass-calibration method for these traces. | DOI:10.1021/bi051297m.s001
|
| NovJ/NovK sample used for supporting gel-filtration traces | has_study_level_assembly_context | Separately reported J2K2 assembly assignment | The main abstract separately reports purification as a J2K2 heterotetramer. This does not identify every SEC peak or establish that the individual SI constructs are the same assayed preparation. | DOI:10.1021/bi051297m |
| Analytical gel filtration of NovJ/NovK samples | has_concentration_labeled_trace | Trace labeled [J], [K] = 155 uM | Figure 1 labels both [J] and [K] as 155 uM, not total tetramer concentration. The plot has early and later absorbance peaks; the red pseudo-Gaussian overlay fits the later peak rather than the entire trace. No exact peak area or fitted retention volume is extracted from the raster. | DOI:10.1021/bi051297m.s001
|
| Trace labeled [J], [K] = 155 uM | constrains_assembly_interpretation | Limits of independent assembly inference | The panels use different plotting scales and provide no fit parameter table, uncertainty or replicate information. These plots alone do not establish a binding constant, unique molecular mass, monomer-versus-aggregate assignment or native in-cell stoichiometry. | DOI:10.1021/bi051297m.s001
|
| Analytical gel filtration of NovJ/NovK samples | has_concentration_labeled_trace | Trace labeled [J], [K] = 22.5 uM | Figure 2 labels both [J] and [K] as 22.5 uM, not total tetramer concentration. The plot has early and later absorbance peaks; the red pseudo-Gaussian overlay fits the later peak rather than the entire trace. No exact peak area or fitted retention volume is extracted from the raster. | DOI:10.1021/bi051297m.s001
|
| Trace labeled [J], [K] = 22.5 uM | constrains_assembly_interpretation | Limits of independent assembly inference | The panels use different plotting scales and provide no fit parameter table, uncertainty or replicate information. These plots alone do not establish a binding constant, unique molecular mass, monomer-versus-aggregate assignment or native in-cell stoichiometry. | DOI:10.1021/bi051297m.s001
|
| Analytical gel filtration of NovJ/NovK samples | has_concentration_labeled_trace | Trace labeled [J], [K] = 2.5 uM | Figure 3 labels both [J] and [K] as 2.5 uM, not total tetramer concentration. The plot has early and later absorbance peaks; the red pseudo-Gaussian overlay fits the later peak rather than the entire trace. No exact peak area or fitted retention volume is extracted from the raster. | DOI:10.1021/bi051297m.s001
|
| Trace labeled [J], [K] = 2.5 uM | constrains_assembly_interpretation | Limits of independent assembly inference | The panels use different plotting scales and provide no fit parameter table, uncertainty or replicate information. These plots alone do not establish a binding constant, unique molecular mass, monomer-versus-aggregate assignment or native in-cell stoichiometry. | DOI:10.1021/bi051297m.s001
|
| Deposited native NovJ sequence reference | is_native_reference_for | NovJ/NovK sample used for supporting gel-filtration traces | UniProtKB:Q9L9F8 is the deposited native sequence reference for NovJ/NovK sample used for supporting gel-filtration traces (node 'sample'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F8 |
| Deposited native NovK sequence reference | is_native_reference_for | NovJ/NovK sample used for supporting gel-filtration traces | UniProtKB:Q9L9F7 is the deposited native sequence reference for NovJ/NovK sample used for supporting gel-filtration traces (node 'sample'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F7 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 10 nodes, 10 edges
Three database-described engineered NovJ variants and subunit/residue annotations, explicitly not primary-panel reanalysis. Review #562. The cathomycin/BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status. Precursor and recombinant protein evidence does not resolve that stereocentre or establish an exact-key merge with fully specified novobiocin. Main-paper evidence is abstract-only; the official four-page supplement and native deposits were inspected directly.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native NovJ reference for database annotations | is_annotated_catalytic_subunit_of | Database-annotated NovJ2K2 complex | UniProt assigns NovJ the catalytic-subunit role in the heterotetramer. This edge cites the database as such; it does not claim a newly inspected isolated-subunit activity assay. | https://www.uniprot.org/uniprotkb/Q9L9F8
|
| Native NovK reference for database annotations | is_annotated_noncatalytic_subunit_of | Database-annotated NovJ2K2 complex | UniProt assigns NovK the non-catalytic-subunit role. That annotation does not mean the protein is dispensable, or establish inactivity in every untested condition. | https://www.uniprot.org/uniprotkb/Q9L9F7
|
| Native NovJ reference for database annotations | is_sequence_reference_for_variant | Database-described engineered NovJ S152A | The Q9L9F8 mutagenesis feature specifies S152A and cites PMID 16171397. The indicated original residue agrees with the 262-aa deposited sequence. Native Q9L9F8 is not assigned as the engineered protein identifier; tag, partner preparation and complete construct remain unresolved. | https://www.uniprot.org/uniprotkb/Q9L9F8 |
| Database-described engineered NovJ S152A | has_database_reported_outcome | Database-reported lower beta-ketotyrosine product formation | The database describes a 2-3-fold decrease in beta-ketotyrosine product formation, not a directly re-extracted kcat or binding affinity. Quantitative assay conditions, loading, replicate count and uncertainty were not independently obtained. | https://www.uniprot.org/uniprotkb/Q9L9F8
|
| Native NovJ reference for database annotations | is_sequence_reference_for_variant | Database-described engineered NovJ Y164F | The Q9L9F8 mutagenesis feature specifies Y164F and cites PMID 16171397. The indicated original residue agrees with the 262-aa deposited sequence. Native Q9L9F8 is not assigned as the engineered protein identifier; tag, partner preparation and complete construct remain unresolved. | https://www.uniprot.org/uniprotkb/Q9L9F8 |
| Database-described engineered NovJ Y164F | has_database_reported_outcome | Database-reported lower catalytic activity | The database describes a 50-fold activity reduction. The primary abstract also mentions a 50-fold effect but does not name the residue, so the Y164F identity remains a database assertion, not an independently read primary panel. Quantitative assay conditions, loading, replicate count and uncertainty were not independently obtained. | https://www.uniprot.org/uniprotkb/Q9L9F8
|
| Native NovJ reference for database annotations | is_sequence_reference_for_variant | Database-described engineered NovJ K168I | The Q9L9F8 mutagenesis feature specifies K168I and cites PMID 16171397. The indicated original residue agrees with the 262-aa deposited sequence. Native Q9L9F8 is not assigned as the engineered protein identifier; tag, partner preparation and complete construct remain unresolved. | https://www.uniprot.org/uniprotkb/Q9L9F8 |
| Database-described engineered NovJ K168I | has_database_reported_outcome | Database-reported unchanged catalytic turnover | The database describes no alteration of catalytic turnover. Without the full primary panel this is not a demonstrated equivalence bound, unchanged substrate affinity or universal absence of a phenotype. Quantitative assay conditions, loading, replicate count and uncertainty were not independently obtained. | https://www.uniprot.org/uniprotkb/Q9L9F8
|
| Native NovJ reference for database annotations | has_annotation_evidence_limits | Database residue annotations without independently inspected primary panels | NovJ Tyr164 proton-acceptor and Ser152 substrate-binding annotations use ECO:0000305; NADP-binding positions 23-26, 73-74 and 164-168 use ECO:0000250. These are not newly observed atomic contacts. The primary mutant panel and a relevant experimental structure were not inspected. | https://www.uniprot.org/uniprotkb/Q9L9F8
|
| Native NovK reference for database annotations | has_annotation_evidence_limits | Database residue annotations without independently inspected primary panels | NovK NADP-binding positions 9-12, 59-60 and 154-158 carry ECO:0000250 similarity evidence. Do not convert them into measured binding constants or structural contacts. | https://www.uniprot.org/uniprotkb/Q9L9F7
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 12 nodes, 12 edges
Native NovM identity, a distinct His8 preparation and bounded primary glycosylation evidence. Review #563. The BGC0000834 cathomycin owner retains its unspecified sugar stereocentre and SEEDED status. These precursor and recombinant assays do not resolve that stereocentre or justify an exact-key merge with fully specified novobiocin.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | contains | Deposited novM locus | AF170880.4 novM spans 14106..15245(+), 1140 nt including stop. | https://mibig.secondarymetabolites.org/go/BGC0000834.5 |
| Deposited novM locus | encodes_native_reference | Deposited native NovM sequence reference | All 379 aa of AAF67506.1 match Q9L9F5 and CDS-aware table-11 translations of AF170880.2/.3/.4. Earlier coordinates are 14054..15193(+). | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited native NovM sequence reference | has_identity_boundary | Historical strain and engineered-sequence limits | The current deposit names S. niveus NCIMB 9219; UniProt taxon 193462 retains historical reference comments including ATCC 23965/NCIMB 11891. These labels do not prove strain synonymy or identify a complete historical fusion. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novM locus | is_named_source_of | Freel Meyers 2003 C-terminal His8 NovM preparation | The abstract reports soluble expression and purification to homogeneity of a C-terminal His8 fusion from S. spheroides. No full fusion sequence or quantitative purity is assigned. | DOI:10.1021/bi0340088
|
| Reported E. coli NovM expression host | expressed | Freel Meyers 2003 C-terminal His8 NovM preparation | The abstract identifies E. coli, not a host strain, vector, yield or induction protocol. | DOI:10.1021/bi0340088
|
| Freel Meyers 2003 C-terminal His8 NovM preparation | catalyzes | NovM precursor noviosylation | Reported turnover exceeds 300 min^-1. Detailed kinetic conditions and uncertainty are outside the inspected abstract. | DOI:10.1021/bi0340088
|
| Novobiocic acid acceptor | is_acceptor_for | NovM precursor noviosylation | The study obtained this aglycon by selective novobiocin degradation. | DOI:10.1021/bi0340088
|
| TDP-L-noviose donor, historical assay label | is_donor_for | NovM precursor noviosylation | The donor came from an eleven-step chemical synthesis beginning with L-rhamnose; this is not an enzymatic sugar-biosynthesis assay. | DOI:10.1021/bi0340088
|
| NovM precursor noviosylation | forms | Desmethyldescarbamoylnovobiocin precursor | The source assigns the glycosylated precursor, not fully tailored novobiocin. | DOI:10.1021/bi0340088
|
| Deposited native NovM sequence reference | has_database_reaction_annotation | Database reaction annotation and donor nomenclature | UniProt gives EC 2.4.1.302 and RHEA:36695: dTDP-4-O-demethyl-beta-L-noviose plus novobiocic acid yields desmethyldescarbamoylnovobiocin, dTDP and H(+). The historical TDP-L-noviose label must not imply an already 4-O-methylated donor. The reaction identifier is not a protein identifier. | https://www.uniprot.org/uniprotkb/Q9L9F5
|
| Database reaction annotation and donor nomenclature | clarifies_historical_label | TDP-L-noviose donor, historical assay label | Retain the source label alongside the database naming; no donor stereochemistry or final-product identity is inferred from the abbreviated name alone. | https://www.uniprot.org/uniprotkb/Q9L9F5 |
| Deposited native NovM sequence reference | has_database_kinetic_annotation | Database-reported NovM kinetic values | UniProt reports Km 32 uM and kcat 313 min^-1 with novobiocic acid. These exact values are database assertions; the primary table and assay conditions were not recovered, and Km is not a measured binding Kd. | https://www.uniprot.org/uniprotkb/Q9L9F5
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 18 nodes, 20 edges
Separate NovM donor/acceptor evidence and abstract-level tandem incubations, without physical-complex claims. Review #563. The BGC0000834 cathomycin owner retains its unspecified sugar stereocentre and SEEDED status. These precursor and recombinant assays do not resolve that stereocentre or justify an exact-key merge with fully specified novobiocin.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Freel Meyers 2003 C-terminal His8 NovM preparation | has_bounded_negative_result | Reported lack of rhamnosyl-transfer activity | The abstract reports no activity with TDP-L-rhamnose; no detection limit or universal rejection of every rhamnose donor is inferred. | DOI:10.1021/bi0340088
|
| TDP-L-rhamnose donor analogue | was_tested_in | Reported lack of rhamnosyl-transfer activity | This donor analogue is distinct from the synthetic TDP-L-noviose used for positive turnover. | DOI:10.1021/bi0340088
|
| TDP-L-rhamnose donor analogue | has_reported_inhibitory_effect | Reported TDP-L-rhamnose inhibition | Reported Ki is 83.5 +/- 5.5 uM. The abstract does not define the uncertainty, replicate count or inhibition model. | DOI:10.1021/bi0340088
|
| Reported TDP-L-rhamnose inhibition | was_measured_with | Freel Meyers 2003 C-terminal His8 NovM preparation | This value belongs to the reported NovM preparation, not native-cell inhibition or antibacterial potency. | DOI:10.1021/bi0340088
|
| Freel Meyers 2003 C-terminal His8 NovM preparation | accepts_reported_alternative_aglycons | Reported alternative coumarin aglycon acceptance | Some coumarin acceptor variants support activity. No uninspected compound list or quantitative ranking is reconstructed. | DOI:10.1021/bi0340088
|
| Albermann 2003 NovM assay preparation | was_tested_in | Albermann aglycon and nucleotide-sugar panel | The primary abstract describes four aglycon analogues and over forty nucleotide sugars; complete construct and individual assay rows remain uninspected. | DOI:10.1021/ol0341086
|
| Albermann aglycon and nucleotide-sugar panel | yielded_reported_products | Three reported new glycosylation products | Three new products are reported. The abstract alone does not establish their exact structures or yields. | DOI:10.1021/ol0341086
|
| Reported alternative coumarin aglycon acceptance | must_be_interpreted_with | Donor-versus-acceptor specificity and preparation limits | Acceptance of selected aglycons does not establish broad sugar-donor promiscuity. | DOI:10.1021/bi0340088
|
| Three reported new glycosylation products | must_be_interpreted_with | Donor-versus-acceptor specificity and preparation limits | Stringency in another panel does not by itself contradict selected aglycon acceptance. No identity between the two engineered preparations is assumed. | DOI:10.1021/ol0341086
|
| Pi 2004 NovL assay preparation | was_used_in | NovL/NovM tandem-incubation assay | NovL and NovM were co-incubated for pathway assembly. | DOI:10.1073/pnas.0403526101
|
| Pi 2004 NovM assay preparation | was_used_in | NovL/NovM tandem-incubation assay | The preparation is study-specific; neither its tag nor full sequence is supplied by the inspected abstract. | DOI:10.1073/pnas.0403526101
|
| NovL/NovM tandem-incubation assay | has_three_enzyme_extension | NovL/NovM/NovP tandem-incubation assay | The reported extension also includes NovP. Co-incubation is not evidence of a stable physical complex. | DOI:10.1073/pnas.0403526101
|
| Pi 2004 NovP assay preparation | was_used_in | NovL/NovM/NovP tandem-incubation assay | NovP is the additional tailoring enzyme, not a demonstrated structural subunit of a NovL-NovM assembly. | DOI:10.1073/pnas.0403526101
|
| NovL/NovM/NovP tandem-incubation assay | was_monitored_by | Time-dependent intermediate and product MS readout | The abstract reports time-dependent MS detection of intermediates and products. New kinetic values, ion assignments and product yields are not asserted from unverified tables or figures. | DOI:10.1073/pnas.0403526101
|
| Time-dependent intermediate and product MS readout | has_evidence_boundary | Donor-versus-acceptor specificity and preparation limits | The 2004 quantitative full-text leads remain uncurated; they must not be silently transferred to either 2003 preparation. | DOI:10.1073/pnas.0403526101
|
| Deposited native NovM sequence reference | is_native_reference_for | Freel Meyers 2003 C-terminal His8 NovM preparation | UniProtKB:Q9L9F5 is the deposited native sequence reference for Freel Meyers 2003 C-terminal His8 NovM preparation (node 'prep'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F5 |
| Deposited native NovM sequence reference | is_native_reference_for | Albermann 2003 NovM assay preparation | UniProtKB:Q9L9F5 is the deposited native sequence reference for Albermann 2003 NovM assay preparation (node 'other_prep'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F5 |
| Deposited native NovM sequence reference | is_native_reference_for | Pi 2004 NovM assay preparation | UniProtKB:Q9L9F5 is the deposited native sequence reference for Pi 2004 NovM assay preparation (node 'm'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F5 |
| Deposited native NovL sequence reference | is_native_reference_for | Pi 2004 NovL assay preparation | UniProtKB:Q9L9F6 is the deposited native sequence reference for Pi 2004 NovL assay preparation (node 'l'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F6 |
| Deposited native NovP sequence reference | is_native_reference_for | Pi 2004 NovP assay preparation | UniProtKB:Q9L9F2 is the deposited native sequence reference for Pi 2004 NovP assay preparation (node 'p'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F2 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 25 nodes, 30 edges
New commercial redox-support proteins and directly inspected early-stage NovO/CouO tests. The supplement does not establish complete sequences/tags for its recombinant preparations. Review #563. The BGC0000834 cathomycin owner retains its unspecified sugar stereocentre and SEEDED status. These precursor and recombinant assays do not resolve that stereocentre or justify an exact-key merge with fully specified novobiocin.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| 2005 supplement holo-NovH preparation | was_used_in | NovH tyrosyl-loading stage | Holo-NovH 30 uM; 75 mM Tris-HCl pH 7.5, ATP 5 mM, MgCl2 10 mM, L-tyrosine 0.5 mM plus [3H]-L-tyrosine 1.8 uM; two hours at room temperature. | DOI:10.1021/bi051599o.s001
|
| NovH tyrosyl-loading stage | prepared_assay_stage | L-Tyrosyl-S-NovH assay intermediate | Carrier-bound precursor, not free tyrosine. | DOI:10.1021/bi051599o.s001
|
| L-Tyrosyl-S-NovH assay intermediate | was_used_in | NovI hydroxylation-stage reconstitution | This stage followed the NovH incubation. | DOI:10.1021/bi051599o.s001
|
| 2005 supplement NovI preparation | was_used_in | NovI hydroxylation-stage reconstitution | NovI 10 uM and NADPH 2 mM; one hour. | DOI:10.1021/bi051599o.s001
|
| Sigma spinach ferredoxin assay reagent | was_supplied_in | NovI hydroxylation-stage reconstitution | Commercial ferredoxin: 10 uM; the source explicitly specifies spinach. | DOI:10.1021/bi051599o.s001
|
| Sigma ferredoxin reductase assay reagent | was_supplied_in | NovI hydroxylation-stage reconstitution | Commercial reductase: 0.1 U. The source does not specify its species, sequence or catalogue number. | DOI:10.1021/bi051599o.s001
|
| NovI hydroxylation-stage reconstitution | prepared_assay_stage | Beta-hydroxytyrosyl-S-NovH assay intermediate | The named stage is beta-hydroxytyrosyl-NovH; no new stereochemical assignment is made. | DOI:10.1021/bi051599o.s001
|
| Beta-hydroxytyrosyl-S-NovH assay intermediate | was_used_in | NovJ/NovK oxidation-stage reconstitution | The NovJ/NovK step followed the NovI incubation. | DOI:10.1021/bi051599o.s001
|
| 2005 supplement NovJ/NovK preparation | was_used_in | NovJ/NovK oxidation-stage reconstitution | NovJ/NovK 10 uM plus NADP(+) 1 mM; one hour. The concentration basis is not specified as subunits or complex. | DOI:10.1021/bi051599o.s001
|
| NovJ/NovK oxidation-stage reconstitution | prepared_assay_stage | Beta-keto-S-NovH assay intermediate | The product-stage label is retained without assuming a complete engineered sequence for its NovH carrier. | DOI:10.1021/bi051599o.s001
|
| 2005 supplement NovO assay preparation | was_tested_in | Stage-specific tethered-precursor methylation tests | NovO 5 uM with SAM 2 mM; one to five hours. | DOI:10.1021/bi051599o.s001
|
| 2005 supplement CouO assay preparation | was_tested_in | Stage-specific tethered-precursor methylation tests | CouO was an alternative methyltransferase at the same reported loading, not an asserted native novobiocin enzyme. | DOI:10.1021/bi051599o.s001
|
| L-Tyrosyl-S-NovH assay intermediate | was_test_substrate_in | Stage-specific tethered-precursor methylation tests | Methyltransferase added after NovH. | DOI:10.1021/bi051599o.s001
|
| Beta-hydroxytyrosyl-S-NovH assay intermediate | was_test_substrate_in | Stage-specific tethered-precursor methylation tests | Methyltransferase added after NovH/NovI. | DOI:10.1021/bi051599o.s001
|
| Beta-keto-S-NovH assay intermediate | was_test_substrate_in | Stage-specific tethered-precursor methylation tests | Methyltransferase added after NovH/NovI/NovJ/NovK. | DOI:10.1021/bi051599o.s001
|
| Stage-specific tethered-precursor methylation tests | was_evaluated_by | Released-analyte RP-HPLC/scintillation readout | Alkaline release from precipitated protein (0.1 M KOH, 60 C, five minutes), followed by RP-HPLC and scintillation counting. | DOI:10.1021/bi051599o.s001
|
| Stage-specific tethered-precursor methylation tests | had_additional_readout | NovH-bound radiolabel autoradiography readout | The autoradiography paragraph names NovO with SAM 1 mM and [3H]-SAM 0.7 uM; SDS gels retained protein-associated label. | DOI:10.1021/bi051599o.s001
|
| ATP-omitted autoradiography comparator | was_compared_in | NovH-bound radiolabel autoradiography readout | The source reports no difference from the otherwise equivalent ATP-omitted reactions. | DOI:10.1021/bi051599o.s001
|
| Released-analyte RP-HPLC/scintillation readout | supports_reported_negative | Reported absence of tethered-precursor methylation | The summary reports no methylation by NovO or CouO at the three tethered stages. | DOI:10.1021/bi051599o.s001
|
| NovH-bound radiolabel autoradiography readout | has_method_scope_limit | Commercial-reagent and method-scope limits | The summary includes both enzymes, but the autoradiography methods specifically name NovO. Do not infer every CouO control or universal substrate impossibility; no detection limit is supplied. | DOI:10.1021/bi051599o.s001
|
| Reported absence of tethered-precursor methylation | has_interpretation_limit | Commercial-reagent and method-scope limits | No new direct-binding, native-flux or structure claim follows from this assay-bounded negative. | DOI:10.1021/bi051599o.s001
|
| Sigma spinach ferredoxin assay reagent | has_identity_and_role_limit | Commercial-reagent and method-scope limits | A supplied reagent is not proof of native partnership, direct binding or omission-tested necessity. No unique UniProt accession is justified. | DOI:10.1021/bi051599o.s001
|
| Sigma ferredoxin reductase assay reagent | has_identity_and_role_limit | Commercial-reagent and method-scope limits | No spinach identity is transferred from the adjacent ferredoxin label to the reductase. Sequence-specific grounding remains unavailable. | DOI:10.1021/bi051599o.s001
|
| 2005 supplement NovO assay preparation | was_tested_in | Free desmethyl-aminocoumarin methylation test | NovO or CouO 20 uM; desmethyl-aminocoumarin 1 mM, Tris-HCl 75 mM pH 8.5, SAM 2 mM, BSA 1 mg/mL, DMSO 10%; overnight. | DOI:10.1021/bi051599o.s001
|
| 2005 supplement CouO assay preparation | was_tested_in | Free desmethyl-aminocoumarin methylation test | CouO was tested as the alternative enzyme, not as a mixed NovO/CouO complex. | DOI:10.1021/bi051599o.s001
|
| Free desmethyl-aminocoumarin methylation test | gave_bounded_negative | No free-aminocoumarin product detected by RP-HPLC | RP-HPLC with authentic 8-methyl-aminocoumarin standard detected no product; no quantified limit of detection is asserted. | DOI:10.1021/bi051599o.s001
|
| Deposited native NovH sequence reference | is_native_reference_for | 2005 supplement holo-NovH preparation | UniProtKB:Q9L9G0 is the deposited native sequence reference for 2005 supplement holo-NovH preparation (node 'h'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9G0 |
| Deposited native NovI sequence reference | is_native_reference_for | 2005 supplement NovI preparation | UniProtKB:Q9L9F9 is the deposited native sequence reference for 2005 supplement NovI preparation (node 'i'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F9 |
| Deposited native NovO sequence reference | is_native_reference_for | 2005 supplement NovO assay preparation | UniProtKB:Q9L9F3 is the deposited native sequence reference for 2005 supplement NovO assay preparation (node 'o'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F3 |
| Deposited native CouO sequence reference | is_native_reference_for | 2005 supplement CouO assay preparation | UniProtKB:Q9F8T9 is the deposited native sequence reference for 2005 supplement CouO assay preparation (node 'couo'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9F8T9 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 18 nodes, 20 edges
Direct 2005 supplement evidence for staged NovM/NovP/NovN assays around NovO, with unresolved product mass and sequence boundaries. Review #563. The BGC0000834 cathomycin owner retains its unspecified sugar stereocentre and SEEDED status. These precursor and recombinant assays do not resolve that stereocentre or justify an exact-key merge with fully specified novobiocin.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| 2005 supplement NovM preparation | was_supplied_in | Ring-C-desmethyl precursor tailoring experiment | NovM 1 uM with TDP-L-noviose 200 uM. | DOI:10.1021/bi051599o.s001
|
| 2005 supplement NovP preparation | was_supplied_in | Ring-C-desmethyl precursor tailoring experiment | NovP 1 uM with SAM 1 mM. | DOI:10.1021/bi051599o.s001
|
| 2005 supplement NovN preparation | was_supplied_in | Ring-C-desmethyl precursor tailoring experiment | NovN 1 uM with ATP 4 mM and carbamoyl phosphate. Separate donor concentration is not unambiguously stated. | DOI:10.1021/bi051599o.s001
|
| 2005 supplement NovO preparation | was_tested_in | Ring-C-desmethyl precursor tailoring experiment | NovO was inserted at the listed stages. Its loading is unspecified here and is not borrowed from the earlier tests. | DOI:10.1021/bi051599o.s001
|
| Ring-C-desmethyl precursor tailoring experiment | has_staged_order | NovM-NovO-NovP-NovN staged incubation | Each enzyme was incubated for twelve hours. Starting mixture: desmethyl-novobiocic acid 6 at 1 mM, Tris 75 mM pH 7.5, SAM 2 mM, BSA 1 mg/mL, DMSO 10%. | DOI:10.1021/bi051599o.s001
|
| NovM-NovO-NovP-NovN staged incubation | contributes_to_study_level_result | No detected C8 methylation after glycosylation | LC-MS evaluation reported no C8 methylation in the glycosylated derivatives. No order-specific numerical conversion is supplied. | DOI:10.1021/bi051599o.s001
|
| Ring-C-desmethyl precursor tailoring experiment | has_staged_order | NovM-NovP-NovO-NovN staged incubation | Each enzyme was incubated for twelve hours. Starting mixture: desmethyl-novobiocic acid 6 at 1 mM, Tris 75 mM pH 7.5, SAM 2 mM, BSA 1 mg/mL, DMSO 10%. | DOI:10.1021/bi051599o.s001
|
| NovM-NovP-NovO-NovN staged incubation | contributes_to_study_level_result | No detected C8 methylation after glycosylation | LC-MS evaluation reported no C8 methylation in the glycosylated derivatives. No order-specific numerical conversion is supplied. | DOI:10.1021/bi051599o.s001
|
| Ring-C-desmethyl precursor tailoring experiment | has_staged_order | NovM-NovP-NovN-NovO staged incubation | Each enzyme was incubated for twelve hours. Starting mixture: desmethyl-novobiocic acid 6 at 1 mM, Tris 75 mM pH 7.5, SAM 2 mM, BSA 1 mg/mL, DMSO 10%. | DOI:10.1021/bi051599o.s001
|
| NovM-NovP-NovN-NovO staged incubation | contributes_to_study_level_result | No detected C8 methylation after glycosylation | LC-MS evaluation reported no C8 methylation in the glycosylated derivatives. No order-specific numerical conversion is supplied. | DOI:10.1021/bi051599o.s001
|
| Ring-C-desmethyl precursor tailoring experiment | has_source_reported_product | Source-labeled NovM,P product | Printed formula C29H33NO10; calculated neutral mass 555.2, observed m/z 554.1 [M-H]-. The product list is study-level, not uniquely assigned to one incubation order. | DOI:10.1021/bi051599o.s001
|
| Ring-C-desmethyl precursor tailoring experiment | has_source_reported_product | Source-labeled NovM,N product | Printed formula C29H32N2O11; calculated neutral mass 580.2, observed m/z 579.2 [M-H]-. The product list is study-level, not uniquely assigned to one incubation order. | DOI:10.1021/bi051599o.s001
|
| Ring-C-desmethyl precursor tailoring experiment | has_source_reported_product | Source-labeled NovM,P,N product | Printed formula C30H34N2O11; calculated neutral mass 598.2, observed m/z 597.2 [M-H]-. The product list is study-level, not uniquely assigned to one incubation order. | DOI:10.1021/bi051599o.s001
|
| Source-labeled NovM,N product | has_identity_conflict | NovM,N formula versus mass discrepancy | Parsing C29H32N2O11 gives nominal neutral mass 584, inconsistent with printed 580.2 and observed 579.2 [M-H]-. Retain all printed values without silently correcting the formula or assigning an exact structure identifier. | DOI:10.1021/bi051599o.s001
|
| No detected C8 methylation after glycosylation | has_interpretation_limit | Staged-assay assignment and construct limits | Ring-C desmethylation is distinct from sugar-O demethylation. These staged experiments neither prove a stable protein complex nor identify a unique physiological enzyme order, final compound stereochemistry or every assay construct. | DOI:10.1021/bi051599o.s001
|
| NovM,N formula versus mass discrepancy | limits_product_assignment | Staged-assay assignment and construct limits | The presence of a NovM,N product in the list does not prove an unlisted NovP-omitted order or failure of NovP in a particular listed incubation. | DOI:10.1021/bi051599o.s001
|
| Deposited native NovM sequence reference | is_native_reference_for | 2005 supplement NovM preparation | UniProtKB:Q9L9F5 is the deposited native sequence reference for 2005 supplement NovM preparation (node 'm'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F5 |
| Deposited native NovP sequence reference | is_native_reference_for | 2005 supplement NovP preparation | UniProtKB:Q9L9F2 is the deposited native sequence reference for 2005 supplement NovP preparation (node 'p'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F2 |
| Deposited native NovN sequence reference | is_native_reference_for | 2005 supplement NovN preparation | UniProtKB:Q9L9F4 is the deposited native sequence reference for 2005 supplement NovN preparation (node 'n'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F4 |
| Deposited native NovO sequence reference | is_native_reference_for | 2005 supplement NovO preparation | UniProtKB:Q9L9F3 is the deposited native sequence reference for 2005 supplement NovO preparation (node 'o'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9F3 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 9 nodes, 11 edges
Three grounded native references and a separate historical NovU sequence; no retrospectively identified assay fusion. Review #564. The BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status; precursor evidence does not resolve that centre or justify an exact-key novobiocin merge.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster | contains | Deposited novW locus | Current CDS 25046..25669(+); historical AF170880.2/.3 coordinates are 24994..25617(+). | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novW locus | encodes_native_reference | Current deposited native novW reference | 207 aa; AAF67516.1; CDS-aware table-11 translation matches the current UniProt sequence exactly. Historical AF170880.2/.3 translations are also exact matches. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Current deposited native novW reference | has_identity_boundary | Native sequence and historical sample boundaries | Current GenBank names S. niveus NCIMB 9219; UniProt retains historical ATCC 23965/NCIMB 11891 reference comments. Sequence identity does not prove strain synonymy or identify every historical tagged sample. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novobiocin cluster | contains | Deposited novU locus | Current CDS 22832..24094(+); historical AF170880.2/.3 coordinates are 22780..24042(+). | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novU locus | encodes_native_reference | Current deposited native novU reference | 420 aa; AAF67514.2; CDS-aware table-11 translation matches the current UniProt sequence exactly. Historical NovU differs at residue 62, as separately represented. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Current deposited native novU reference | has_identity_boundary | Native sequence and historical sample boundaries | Current GenBank names S. niveus NCIMB 9219; UniProt retains historical ATCC 23965/NCIMB 11891 reference comments. Sequence identity does not prove strain synonymy or identify every historical tagged sample. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novobiocin cluster | contains | Deposited novS locus | Current CDS 20870..21736(+); historical AF170880.2/.3 coordinates are 20818..21684(+). | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novS locus | encodes_native_reference | Current deposited native novS reference | 288 aa; AAF67512.1; CDS-aware table-11 translation matches the current UniProt sequence exactly. Historical AF170880.2/.3 translations are also exact matches. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Current deposited native novS reference | has_identity_boundary | Native sequence and historical sample boundaries | Current GenBank names S. niveus NCIMB 9219; UniProt retains historical ATCC 23965/NCIMB 11891 reference comments. Sequence identity does not prove strain synonymy or identify every historical tagged sample. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novU locus | has_historical_translation | Historical deposited NovU Val62 sequence | AF170880.2 and .3 both encode 420-aa AAF67514.1; it differs from current Q9L9E7/AAF67514.2 only at Val62 versus Leu62. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.2 |
| Historical deposited NovU Val62 sequence | must_not_be_conflated_with | Current deposited native novU reference | The historical Val62 reference is not the current Leu62 sequence. Experimental allele identity remains unresolved without the complete construct sequence; this difference is not an engineered mutant claim. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 18 nodes, 21 edges
Primary abstract-level NovW/NovU/NovS reactions, with unresolved product nomenclature and pathway order. Review #564. The BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status; precursor evidence does not resolve that centre or justify an exact-key novobiocin merge.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Reported E. coli expression host | expressed | Thuy 2005 NovW assay preparation | The abstract identifies the host species but does not supply a full fusion sequence or a strain-specific preparation. | DOI:10.1016/j.abb.2005.01.012
|
| Reported E. coli expression host | expressed | Thuy 2005 NovU assay preparation | The abstract identifies the host species but does not supply a full fusion sequence or a strain-specific preparation. | DOI:10.1016/j.abb.2005.01.012
|
| Reported E. coli expression host | expressed | Thuy 2005 NovS assay preparation | The abstract identifies the host species but does not supply a full fusion sequence or a strain-specific preparation. | DOI:10.1016/j.abb.2005.01.012
|
| Thuy 2005 NovW assay preparation | was_used_in | Reported joint NovW/NovU conversion | NovW and NovU jointly yielded the named methylated precursor. | DOI:10.1016/j.abb.2005.01.012
|
| Thuy 2005 NovU assay preparation | was_used_in | Reported joint NovW/NovU conversion | Joint enzymatic activity does not demonstrate physical association. | DOI:10.1016/j.abb.2005.01.012
|
| dTDP-4-keto-6-deoxy-D-glucose | was_supplied_to | Reported joint NovW/NovU conversion | The named starting sugar is retained without assigning an unverified structure identifier. | DOI:10.1016/j.abb.2005.01.012
|
| Reported joint NovW/NovU conversion | formed_reported_product | Source-assigned dTDP-4-keto-6-deoxy-5-C-methyl-L-lyxo-hexose | Source-assigned sugar intermediate, not mature antibiotic. | DOI:10.1016/j.abb.2005.01.012
|
| Thuy 2005 NovW assay preparation | was_used_in | Reported NovW-only precursor treatment | This NovW-only treatment is distinct from joint NovW/NovU turnover. | DOI:10.1016/j.abb.2005.01.012
|
| dTDP-4-keto-6-deoxy-D-glucose | was_supplied_to | Reported NovW-only precursor treatment | The abstract names the same starting sugar. | DOI:10.1016/j.abb.2005.01.012
|
| Reported NovW-only precursor treatment | supplied | NovW-treated substrate supplied to NovS | No exact product stereochemistry is added from the abstract alone. | DOI:10.1016/j.abb.2005.01.012
|
| NovW-treated substrate supplied to NovS | was_supplied_to | Reported NADH-dependent NovS reduction | The NovS experiment follows the NovW-only treatment. | DOI:10.1016/j.abb.2005.01.012
|
| Thuy 2005 NovS assay preparation | was_used_in | Reported NADH-dependent NovS reduction | NovS reduced the supplied NovW-treated material. | DOI:10.1016/j.abb.2005.01.012
|
| NADH assay reagent | was_supplied_to | Reported NADH-dependent NovS reduction | NADH is reported, not NADPH. | DOI:10.1016/j.abb.2005.01.012
|
| Reported NADH-dependent NovS reduction | formed_reported_product | Abstract-assigned dTDP-L-rhamnose | Retain the primary abstract product name. | DOI:10.1016/j.abb.2005.01.012
|
| Abstract-assigned dTDP-L-rhamnose | has_annotation_conflict | NovS primary/database product discrepancy | UniProt NovS instead names dTDP-5-methyl-L-rhamnose for the NovW-derived substrate. Neither the methyl substituent nor a corrected chemical identifier is inferred here. | DOI:10.1016/j.abb.2005.01.012 |
| Reported joint NovW/NovU conversion | has_ordering_limit | Unresolved NovW/NovU physiological ordering | Tello Figure 2 proposes both W-before-U and U-before-W routes. The caption does not establish one physiological order. | DOI:10.1039/B515763C
|
| Unresolved NovW/NovU physiological ordering | limits_interpretation | Abstract-level preparation and reaction limits | No ordered native pathway or stable NovW/NovU/NovS complex follows from these data. | DOI:10.1039/B515763C
|
| NovS primary/database product discrepancy | limits_interpretation | Abstract-level preparation and reaction limits | The full 2005 paper remains uninspected; do not transfer unverified kinetic values, cofactor stoichiometry or construct details. | DOI:10.1016/j.abb.2005.01.012
|
| Deposited native NovW sequence reference | is_native_reference_for | Thuy 2005 NovW assay preparation | UniProtKB:Q9L9E5 is the deposited native sequence reference for Thuy 2005 NovW assay preparation (node 'w'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9E5 |
| Deposited native NovU sequence reference | is_native_reference_for | Thuy 2005 NovU assay preparation | UniProtKB:Q9L9E7 is the deposited native sequence reference for Thuy 2005 NovU assay preparation (node 'u'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9E7 |
| Deposited native NovS sequence reference | is_native_reference_for | Thuy 2005 NovS assay preparation | UniProtKB:Q9L9E9 is the deposited native sequence reference for Thuy 2005 NovS assay preparation (node 's'); the experimental preparation node remains ungrounded because recombinant tags, host-specific processing, or undeposited construct sequences are not equated with the native entry. | https://www.uniprot.org/uniprotkb/Q9L9E9 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 20 nodes, 22 edges
New support proteins, source-reported kinetics and solvent-exchange evidence with explicit primary-text and database limits. Review #564. The BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status; precursor evidence does not resolve that centre or justify an exact-key novobiocin merge.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native NovW sequence reference | is_native_reference_for | Tello N-terminal His-tagged NovW preparation | The preparation is N-His NovW from pET-15b, not native Q9L9E5 itself. No full experimental fusion sequence or identity with the scar-bearing 2C0Z entity is established. | DOI:10.1039/B515763C |
| E. coli BL21(DE3) expression host | expressed | Tello N-terminal His-tagged NovW preparation | Ni-NTA then SEC purification; reported yield about 13 mg/L, not assay loading. | DOI:10.1039/B515763C
|
| Reported GroES coexpression partner | was_coexpressed_with | Tello N-terminal His-tagged NovW preparation | Reported production partner; sequence-specific grounding is unavailable. | DOI:10.1039/B515763C
|
| Reported GroEL coexpression partner | was_coexpressed_with | Tello N-terminal His-tagged NovW preparation | No omission-tested necessity or native biosynthetic subunit is asserted. | DOI:10.1039/B515763C
|
| Substrate-preparation RmlB reagent | was_used_in | Enzymatic assay-substrate preparation | RmlB 1.5 mg; dTDP-glucose 10 mg; NAD(+) 1 mM; overnight at 37 C. | DOI:10.1039/B515763C
|
| Enzymatic assay-substrate preparation | supplied | dTDP-6-deoxy-D-xylo-4-hexulose assay substrate | Proton NMR was used for purity checking; no numerical purity is supplied. | DOI:10.1039/B515763C
|
| dTDP-6-deoxy-D-xylo-4-hexulose assay substrate | was_used_in | NADPH-coupled double-epimerase assay | The readout couples double-epimerized product reduction to NADPH consumption. | DOI:10.1039/B515763C
|
| Coupling-assay RmlD reagent | was_supplied_in | NADPH-coupled double-epimerase assay | RmlD is the coupling reductase, not a demonstrated native NovW partner. | DOI:10.1039/B515763C
|
| Tello N-terminal His-tagged NovW preparation | was_tested_in | NADPH-coupled double-epimerase assay | Coupled assay, not a direct binding or native-flux measurement. | DOI:10.1039/B515763C
|
| NADPH-coupled double-epimerase assay | yielded_reported_fit | NovW coupled-assay kinetic estimates | Table 2 text: Km 0.97 +/- 0.01 mM; kcat 0.020 +/- 0.001 s^-1; kcat/Km 0.021 s^-1 mM^-1. Uncertainty type, replicate count and full referenced protocol remain unresolved; table pixels were unavailable. | DOI:10.1039/B515763C
|
| NovW coupled-assay kinetic estimates | was_compared_in | Cross-study kinetic comparator values | Nonzero double-epimerase turnover is retained; Km is not a binding Kd and these values are not specific 3-epimerase kinetics. | DOI:10.1039/B515763C
|
| Literature EvaD kinetic comparator | has_literature_comparator_row | Cross-study kinetic comparator values | Cited EvaD row: Km 0.27 +/- 0.03 mM; kcat 0.08 +/- 0.01 s^-1; efficiency 0.33 s^-1 mM^-1. Source reference 12, not a new measurement on the NovW preparation. | DOI:10.1039/B515763C
|
| Literature RmlC kinetic comparator | has_literature_comparator_row | Cross-study kinetic comparator values | Cited S. enterica RmlC row: Km 0.71 +/- 0.03 mM; kcat 39 +/- 6.6 s^-1; efficiency 54.9 s^-1 mM^-1. Source reference 17; preserve printed values without inventing a shared uncertainty model or identity with the 2006 isotope-assay preparation. | DOI:10.1039/B515763C
|
| Tello N-terminal His-tagged NovW preparation | was_tested_in | Solvent-deuterium incorporation assay | NovW was incubated with substrate in D2O. | DOI:10.1039/B515763C
|
| dTDP-6-deoxy-D-xylo-4-hexulose assay substrate | was_used_in | Solvent-deuterium incorporation assay | Solvent exchange was monitored on substrate, not a demonstrated isolated physiological product. | DOI:10.1039/B515763C
|
| Tello isotope-assay RmlC comparator preparation | was_comparator_in | Solvent-deuterium incorporation assay | The text reports parallel equimolar RmlC; do not identify an exact accession from the comparator name alone. | DOI:10.1039/B515763C
|
| Solvent-deuterium incorporation assay | yielded_reported_readout | Workup-dependent GC-MS isotope readout | Reduction/cleavage/reduction/acetylation preceded GC-MS. Text assigns 6.57/6.65-min derivatives and ions 218/232 for NovW versus 218/233 for RmlC. Figure 4 images were not independently inspected. | DOI:10.1039/B515763C
|
| Workup-dependent GC-MS isotope readout | supports_bounded_assignment | Bounded NovW 3-epimerase functional revision | The reported labeling supports position 3 exchange for NovW versus positions 3 and 5 for RmlC under the tested conditions, not universal absence of any position 5 chemistry. | DOI:10.1039/B515763C
|
| NovW coupled-assay kinetic estimates | supports_bounded_assignment | Bounded NovW 3-epimerase functional revision | The conclusion retains chemical competence for double epimerization while assigning kinetically significant 3-epimerase activity. | DOI:10.1039/B515763C
|
| Bounded NovW 3-epimerase functional revision | has_database_scope_conflict | In-vitro experiment versus in-vivo database wording | UniProt CAUTION calls the later result in vivo; the primary paper describes recombinant in-vitro kinetic/isotope assays. The graph does not repeat the in-vivo claim as experiment. | https://www.uniprot.org/uniprotkb/Q9L9E5 |
| Cross-study kinetic comparator values | has_interpretation_limit | Assay/support-protein and uncertainty limits | Comparator rows originate in other papers. Full RmlB/RmlC/RmlD/EvaD construct sequences and matched assay conditions were not established here. | DOI:10.1039/B515763C
|
| In-vitro experiment versus in-vivo database wording | has_interpretation_limit | Assay/support-protein and uncertainty limits | Family similarity and a single structural conformation do not uniquely determine catalytic regiospecificity. | DOI:10.1039/B515763C
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 13 nodes, 16 edges
Scar-bearing 2C0Z identity, coordinate and assembly evidence, plus separately scoped preliminary solution-size evidence. Review #564. The BGC0000834 owner retains its unspecified sugar stereocentre and SEEDED status; precursor evidence does not resolve that centre or justify an exact-key novobiocin merge.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native NovW sequence reference | matches_core_of | 2C0Z scar-bearing NovW protein entity | Deposited entity: SGLVPRGSH plus the exact 207-aa native sequence, 216 aa total. The extension means the entire entity is not native Q9L9E5; no specific processing mechanism is inferred. | https://files.rcsb.org/download/2C0Z.cif |
| Deposited BL21(DE3) GOLD expression host | is_deposited_host_for | 2C0Z scar-bearing NovW protein entity | Deposit names BL21(DE3), variant GOLD, pET-15B; these metadata are not transferred to another assay preparation. | https://files.rcsb.org/download/2C0Z.cif
|
| Deposited 2C0Z X-ray structure | models | 2C0Z scar-bearing NovW protein entity | X-ray resolution 1.60 A; Rwork 0.150 and Rfree 0.195. Refinement statistics do not validate every mechanistic interpretation. | https://files.rcsb.org/download/2C0Z.cif
|
| 2C0Z scar-bearing NovW protein entity | has_coordinate_coverage | Observed residues and unobserved C terminus | One polymer chain A contains 199 modeled residues: extension -8..0 and native 1..190. Native 191..207 are unobserved, not a demonstrated engineered deletion. | https://files.rcsb.org/download/2C0Z.cif |
| Deposited 2C0Z X-ray structure | has_deposited_assembly | Deposited author/PISA dimeric assembly | Assembly 1 is author/PISA dimeric, generated by operators 1 and 2 from one asymmetric-unit chain. This is not by itself a solution stoichiometry measurement. | https://files.rcsb.org/download/2C0Z.cif
|
| Deposited 2C0Z X-ray structure | contains_modeled_ligands | Deposited sulfate and ethylene glycol ligands | Asymmetric unit: one SO4 and three EDO molecules; no nucleotide-sugar ligand is present. | https://files.rcsb.org/download/2C0Z.cif
|
| Deposited sulfate and ethylene glycol ligands | has_coordinate_proximity | Ser165 to sulfate coordinate proximity | Chain A Ser165 OG to sulfate A1191 O4 is 2.8405 A in deposited coordinates; not a binding constant or catalytic intermediate. | https://files.rcsb.org/download/2C0Z.cif
|
| Ser165 to sulfate coordinate proximity | is_local_to | 2C0Z scar-bearing NovW protein entity | This proximity does not identify a physiological substrate contact or measure interaction strength. | https://files.rcsb.org/download/2C0Z.cif
|
| Deposited 2C0Z X-ray structure | has_unresolved_contact_claim | Unverifiable Arg59 terminal-group contact | The Manchester excerpt of DOI:10.1002/prot.20818 describes Arg59 contacting sulfate. Current 2C0Z explicitly omits Arg59 NE/CZ/NH1/NH2, preventing direct measurement of a guanidinium contact. No missing atom coordinates are reconstructed. | https://research.manchester.ac.uk/en/publications/the-16-%C3%A5-resolution-crystal-structure-of-novw-a-4-keto-6-deoxy-su/ |
| Deposited 2C0Z X-ray structure | contains_conserved_positions | Conserved candidate catalytic residues | Native and deposit numbering agree for His62, Lys72, Tyr132 and Asp168. Conservation and structure alone do not prove residue-specific chemistry; no NovW mutant measurements are asserted from this comparison. | https://files.rcsb.org/download/2C0Z.cif |
| Native NovW sequence reference | is_gene_reference_for | Jakimowicz 2003 crystallization preparation | The 2003 abstract names recombinant NovW but does not establish the complete experimental sequence or equality with the later deposited entity. | DOI:10.1107/S0907444903012848
|
| Jakimowicz 2003 crystallization preparation | has_reported_solution_size_inference | Abstract-reported NovW DLS dimer inference | The abstract reports DLS suggesting a dimer, separately from a monomeric asymmetric unit and 2.0-A preliminary diffraction. DLS conditions and numerical mass estimates were not inspected. | DOI:10.1107/S0907444903012848
|
| Abstract-reported NovW DLS dimer inference | must_be_kept_distinct_from | Deposited author/PISA dimeric assembly | Preparation-specific DLS inference and author/PISA assembly agree qualitatively but are not the same measurement or proof of identical samples. | DOI:10.1107/S0907444903012848 |
| Unverifiable Arg59 terminal-group contact | limits_interpretation | Structure, solution and historical-source boundaries | An institutional excerpt and a current incomplete side chain are not reconciled by inventing an earlier coordinate version or calling an uninspected model experimental. | https://files.rcsb.org/download/2C0Z.cif
|
| Conserved candidate catalytic residues | limits_interpretation | Structure, solution and historical-source boundaries | Observed sulfate and conserved residues do not establish nucleotide-sugar binding geometry, proton transfer or a unique physiological enzyme order. | https://files.rcsb.org/download/2C0Z.cif
|
| 2C0Z scar-bearing NovW protein entity | has_source_metadata_boundary | Structure, solution and historical-source boundaries | 2C0Z retains S. niveus NCIMB 11891, taxon 1352941; current AF170880.4 names NCIMB 9219. Exact protein-core agreement does not erase the strain discrepancy. | https://files.rcsb.org/download/2C0Z.cif |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 9 nodes, 9 edges
Native novE cassette genetics, residual product and unresolved polarity/rescue limits. Partial biosynthesis curation (review #589), not full-record sign-off. The cathomycin owner retains its unspecified sugar stereocentre; source-assigned novobiocin products remain ungrounded, not exact-key merged with CID 54675769. Native deposited sequences do not retrospectively identify every historical strain stock, complete preparation or engineered allele.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | encodes_native_reference | Native deposited NovE regulatory-protein reference | AF170880.4 CDS 4859..5512(+), 1-based inclusive, encodes AAF67498.1, 217 aa. CDS-aware table-11 translations across AF170880.2/.3/.4 exactly match the selected UniProt sequence. Old .2/.3 qualifiers are S. caeruleus, taxon 195949, NCIB 11891; .4 is S. niveus, taxon 193462, NCIMB 9219. The published correction assigns the deposit to 9219, not 11891. The thesis additionally prints 11897 in its strain table versus 11891 in results. These are retained source conflicts, not proof that all experimental stocks were identical. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Native novE replacement in cosmid 10-9C-E-773 | replaces_coding_locus_of | Native deposited NovE regulatory-protein reference | Gene-matched native reference only, not the sequence of an intact mutant protein. The ApraR/oriT cassette replacement is distinct from the later heterologous 81-nt-scar deletion. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1 |
| Native novE replacement in cosmid 10-9C-E-773 | was_introduced_in | Historical S. spheroides AE-E2, AE-E3, AE-E4 mutants | Apramycin-resistant, kanamycin-sensitive double-crossover clones; Southern BamHI comparison 962 versus 2296 bp. Three independent mutants were analyzed. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Historical S. spheroides AE-E2, AE-E3, AE-E4 mutants | retained_reported_production_of | Source-assigned residual novobiocin | All three mutants retained product assigned using HPLC and comparison with an authentic standard by 1H NMR and negative-ion FAB MS. Not absolute zero or a new complete stereochemical determination. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1 |
| Historical S. spheroides AE-E2, AE-E3, AE-E4 mutants | has_production_observation | Native mutant and wild-type HPLC production comparison | Source average 1.5 mg/L in the three mutants; the passage does not give per-clone titers, uncertainty or a detection limit. HPLC methods are in PDF pages 63-64. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Historical S. spheroides wild-type comparator | provides_production_comparator | Native mutant and wild-type HPLC production comparison | Source wild-type 37 mg/L; same native-study comparison, not the heterologous M512 20 mg/L baseline. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Native mutant and wild-type HPLC production comparison | supports_regulatory_interpretation | Proposed positive regulatory role of NovE | Retained product argues against an essential catalytic step under these conditions. Possible cassette polar effects remain; no direct binding or NovE catalytic reaction is established. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1 |
| Native deposited NovE regulatory-protein reference | has_proposed_pathway_role | Proposed positive regulatory role of NovE | Regulatory inference is bounded by the native-mutant experiment, not a physical NovE-NovG interaction. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Historical S. spheroides AE-E2, AE-E3, AE-E4 mutants | was_subject_of_attempted_rescue | Attempted pAE-E4 native-mutant complementation | pAE-E4 plasmid presence could not be demonstrated. This technical failure is not biological non-rescue, and later heterologous complementation does not retrospectively resolve it. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 9 nodes, 11 edges
Heterologous NovG genetics with source-specific residual production and partial rescue. Partial biosynthesis curation (review #589), not full-record sign-off. The cathomycin owner retains its unspecified sugar stereocentre; source-assigned novobiocin products remain ungrounded, not exact-key merged with CID 54675769. Native deposited sequences do not retrospectively identify every historical strain stock, complete preparation or engineered allele.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | encodes_native_reference | Native deposited NovG regulatory-protein reference | AF170880.4 CDS 6782..7738(+), 1-based inclusive, encodes AAF67500.1, 318 aa. CDS-aware table-11 translations across AF170880.2/.3/.4 exactly match the selected UniProt sequence. Old .2/.3 qualifiers are S. caeruleus, taxon 195949, NCIB 11891; .4 is S. niveus, taxon 193462, NCIMB 9219. The published correction assigns the deposit to 9219, not 11891. The thesis additionally prints 11897 in its strain table versus 11891 in results. These are retained source conflicts, not proof that all experimental stocks were identical. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| nov-AE10 novG scar-deletion construct | deletes_coding_locus_of | Native deposited NovG regulatory-protein reference | Replacement cassette removed by XbaI/SpeI digestion and religation, leaving a reported 18 nt scar between start/stop. Not a native NovG protein sequence or the later 81 nt NovE allele. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| nov-AE10 novG scar-deletion construct | was_integrated_in | S. coelicolor M512 nov-AE10 plus empty pWHM3 | Site-specific integration in M512; PstI Southern 8.1 versus 7.2 kb distinguishes intact and deletion loci. Complete altered chromosome sequence was not established. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 nov-AE10 plus empty pWHM3 | retained_reduced_production_of | Source-assigned novobiocin in heterologous cultures | 0.4 mg/L, 2% of the intact comparator, not zero. Residual-product assignment by negative-ion FAB MS, m/z 611, compared with authentic standard. HPLC culture-supernatant production, 305 nm and authentic standard; Table III.5 means 2-4 independent mutants with two determinations each, without row-specific n or error bars. Multicopy-production culture 30 C, 200 rpm, 7 days in CDM, no antibiotics. No exact owner-stereocentre assignment is inferred. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1 |
| S. coelicolor M512 nov-BG1 plus empty pWHM3 | has_comparator_production_of | Source-assigned novobiocin in heterologous cultures | 20 mg/L, 100%, with empty pWHM3. HPLC culture-supernatant production, 305 nm and authentic standard; Table III.5 means 2-4 independent mutants with two determinations each, without row-specific n or error bars. Multicopy-production culture 30 C, 200 rpm, 7 days in CDM, no antibiotics. No exact owner-stereocentre assignment is inferred. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 nov-AE10 plus empty pWHM3 | is_background_for_complementation | nov-AE10 background plus pAE8 NovG expression plasmid | pAE8 contains novG with 336 bp upstream in promoterless replicative pWHM3; full vector or preparation sequence identity is not claimed. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| nov-AE10 background plus pAE8 NovG expression plasmid | partially_restored_production_of | Source-assigned novobiocin in heterologous cultures | 16 mg/L, reported 79%, not silently recalculated as 80% from rounded titers. This supports complementation but is not full 100% restoration. HPLC culture-supernatant production, 305 nm and authentic standard; Table III.5 means 2-4 independent mutants with two determinations each, without row-specific n or error bars. Multicopy-production culture 30 C, 200 rpm, 7 days in CDM, no antibiotics. No exact owner-stereocentre assignment is inferred. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 nov-BG1 plus empty pWHM3 | is_background_for_overexpression | Intact nov-BG1 background plus pAE8 | Same pAE8 introduced into the intact-cluster host, not a deletion rescue. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Intact nov-BG1 background plus pAE8 | increased_reported_production_of | Source-assigned novobiocin in heterologous cultures | 54 mg/L, 270% of the intact empty-vector comparator. This is product titer, not measured NovG protein abundance or a kinetic parameter. HPLC culture-supernatant production, 305 nm and authentic standard; Table III.5 means 2-4 independent mutants with two determinations each, without row-specific n or error bars. Multicopy-production culture 30 C, 200 rpm, 7 days in CDM, no antibiotics. No exact owner-stereocentre assignment is inferred. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| nov-AE10 background plus pAE8 NovG expression plasmid | supports_regulatory_interpretation | Positive NovG contribution to production in the tested host | The genetic rescue and overexpression support a positive role under tested conditions. The source argues against polarity; no independent measurement excludes every polar effect. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1 |
| Native deposited NovG regulatory-protein reference | has_supported_regulatory_role | Positive NovG contribution to production in the tested host | No biosynthetic catalytic activity or physical protein complex follows from these phenotypes. Direct DNA-fragment binding is a separate experiment and graph. | DOI:10.1099/mic.0.27669-0
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 11 edges
Purified NovG fragment-binding evidence, separated from genetic regulation and motif prediction. Partial biosynthesis curation (review #589), not full-record sign-off. The cathomycin owner retains its unspecified sugar stereocentre; source-assigned novobiocin products remain ungrounded, not exact-key merged with CID 54675769. Native deposited sequences do not retrospectively identify every historical strain stock, complete preparation or engineered allele.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | encodes_native_reference | Native deposited NovG regulatory-protein reference | AF170880.4 CDS 6782..7738(+), 1-based inclusive, encodes AAF67500.1, 318 aa. CDS-aware table-11 translations across AF170880.2/.3/.4 exactly match the selected UniProt sequence. Old .2/.3 qualifiers are S. caeruleus, taxon 195949, NCIB 11891; .4 is S. niveus, taxon 193462, NCIMB 9219. The published correction assigns the deposit to 9219, not 11891. The thesis additionally prints 11897 in its strain table versus 11891 in results. These are retained source conflicts, not proof that all experimental stocks were identical. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Native deposited NovG regulatory-protein reference | is_native_sequence_reference_for | pAE-G5-derived N-terminal His6 NovG preparation | Gene-matched native reference, not an exact full His6 fusion, independently sequenced plasmid or sample identity. The pAE-G5 insert was prepared through pAE-G4 from nov-BG1; full vector junction reconstruction was not performed. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| E. coli BL21(DE3)pLysS expression host | produced_tagged_preparation | pAE-G5-derived N-terminal His6 NovG preparation | pRSET B expression construct pAE-G5; soluble preparation obtained with 0.5 mM IPTG at 30 C for 1.5 h. Longer-induction insolubility does not establish a universal expression limit. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| pAE-G5-derived N-terminal His6 NovG preparation | has_purification_readout | Nickel-affinity eluate and SDS-PAGE observation | Nickel-affinity eluate gives an approximately 40 kDa SDS-PAGE band versus calculated 38.1 kDa. About 15 ug/g fresh cells is an SDS-based recovery estimate, not purity percent or catalytic activity. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| pAE-G5-derived N-terminal His6 NovG preparation | was_tested_in | His6 NovG gel mobility-shift assay | About 4 ng DIG-end-labelled DNA and 0.5 ug protein in a 20 uL assay, 25 C for 15 min, followed by native gel analysis. These are the older thesis conditions, not the later NovE/NovG protein-load comparison. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1 |
| His6 NovG gel mobility-shift assay | showed_mobility_shift_for | Tested novG-novH DNA fragment 6 | Fragment 6 in Figure III.17, reported 209 bp. No Kd, kinetic constant or binding stoichiometry is inferred from the gel. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| His6 NovG gel mobility-shift assay | showed_mobility_shift_for | Tested cloG-cloY DNA fragment 1 | Fragment 1 in Figure III.18, reported 235 bp. This is NovG binding heterologous clo DNA, not identification of a clorobiocin regulator protein. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| His6 NovG gel mobility-shift assay | did_not_show_shift_for | Other tested nov and clo DNA fragments | Other Figure III.17 nov fragments, including the second tested novG-novH-region fragment, and the two Figure III.18 cloH-upstream fragments did not shift under tested conditions. This is not universal nonbinding or a genome-wide specificity result. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Specific plasmid versus empty-vector competition | reduced_observed_shift_in | His6 NovG gel mobility-shift assay | Approximately 125-fold molar excess of pAE7 or pAE9 strongly reduced the corresponding shift; matched empty pBluescript SK(-) or pSPORT1 controls did not significantly change it. No quantified occupancy or affinity ratio was reported here. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Tested novG-novH DNA fragment 6 | contributed_to_predicted_motif | Sequence-inferred candidate NovG recognition motif | Sequence comparison with the clo fragment and established StrR sites suggested conserved inverted repeats. This is in silico motif inference, not a NovG DNase I footprint. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Tested cloG-cloY DNA fragment 1 | contributed_to_predicted_motif | Sequence-inferred candidate NovG recognition motif | The cited footprinting experiment was for StrR, not this NovG preparation. No experimentally tested couG-couY binding or physical protein complex follows. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 12 nodes, 16 edges
Later heterologous NovE deletion, overexpression and reciprocal regulatory-gene rescue, with source-specific titers and partial-complementation limits. Partial biosynthesis curation (review #589), not full-record sign-off. The cathomycin owner retains its unspecified sugar stereocentre; source-assigned novobiocin products remain ungrounded, not exact-key merged with CID 54675769. Native deposited sequences do not retrospectively identify every historical strain stock, complete preparation or engineered allele.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | encodes_native_reference | Native deposited NovE regulatory-protein reference | AF170880.4 CDS 4859..5512(+), 1-based inclusive, encodes AAF67498.1, 217 aa. CDS-aware table-11 translations across AF170880.2/.3/.4 exactly match the selected UniProt sequence. Old .2/.3 qualifiers are S. caeruleus, taxon 195949, NCIB 11891; .4 is S. niveus, taxon 193462, NCIMB 9219. The published correction assigns the deposit to 9219, not 11891. The thesis additionally prints 11897 in its strain table versus 11891 in results. These are retained source conflicts, not proof that all experimental stocks were identical. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novobiocin cluster underlying the cathomycin owner | encodes_native_reference | Native deposited NovG regulatory-protein reference | AF170880.4 CDS 6782..7738(+), 1-based inclusive, encodes AAF67500.1, 318 aa. CDS-aware table-11 translations across AF170880.2/.3/.4 exactly match the selected UniProt sequence. Old .2/.3 qualifiers are S. caeruleus, taxon 195949, NCIB 11891; .4 is S. niveus, taxon 193462, NCIMB 9219. The published correction assigns the deposit to 9219, not 11891. The thesis additionally prints 11897 in its strain table versus 11891 in results. These are retained source conflicts, not proof that all experimental stocks were identical. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| nov-VD2 novE scar-deletion construct | deletes_coding_locus_of | Native deposited NovE regulatory-protein reference | nov-VD1 cassette replacement followed by FLP excision gives a reported 81 nt scar. Distinct from the native NovE cassette and 18 nt NovG scar. The thesis's pUG019 FRT description differs from older XbaI/SpeI cassette descriptions; complete construct identity unverified. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1 |
| nov-VD2 novE scar-deletion construct | was_integrated_in | M512 nov-VD2 plus empty pWHM3 | Site-specific integration and PstI Southern comparison support the altered cluster; no complete chromosome or deletion-product protein sequence is asserted. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| M512 nov-VD2 plus empty pWHM3 | retained_reduced_production_of | Source-assigned novobiocin in later heterologous cultures | 0.14 mg/L, 0.7% of the empty-vector intact-cluster comparator, not zero. HPLC supernatant analysis at 305 nm against a standard; CDM flask cultures, 30 C, 200 rpm, seven days without antibiotics. Table III.1 means at least two independent mutants; row-specific n and uncertainty not given. Not the later deep-well qPCR baseline. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| M512 nov-BG1 plus empty pWHM3 | has_comparator_production_of | Source-assigned novobiocin in later heterologous cultures | 20.0 mg/L, 100%, with empty pWHM3; the no-extra-plasmid row is separately 19.3 mg/L. HPLC supernatant analysis at 305 nm against a standard; CDM flask cultures, 30 C, 200 rpm, seven days without antibiotics. Table III.1 means at least two independent mutants; row-specific n and uncertainty not given. Not the later deep-well qPCR baseline. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| M512 nov-VD2 plus empty pWHM3 | is_background_for_complementation | nov-VD2 background plus NovE expression plasmid pVD4 | pVD4 carries novE plus reported 599 bp upstream in promoterless pWHM3. The primer-defined native interval and source insert-table endpoint differ by one nucleotide; full plasmid identity was not independently established. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| nov-VD2 background plus NovE expression plasmid pVD4 | partially_restored_production_of | Source-assigned novobiocin in later heterologous cultures | Narrative reports up to 70% of intact-cluster production, not a table mean or measured 70% in every clone. This is heterologous rescue, not resolution of the earlier native pAE-E4 technical failure. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1 |
| M512 nov-BG1 plus empty pWHM3 | is_background_for_overexpression | Intact nov-BG1 background plus pVD4 | pVD4 added to the intact-cluster host, separate from deletion rescue. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Intact nov-BG1 background plus pVD4 | increased_reported_production_of | Source-assigned novobiocin in later heterologous cultures | 38.1 mg/L, 191% of the intact empty-vector comparator. HPLC supernatant analysis at 305 nm against a standard; CDM flask cultures, 30 C, 200 rpm, seven days without antibiotics. Table III.1 means at least two independent mutants; row-specific n and uncertainty not given. Not the later deep-well qPCR baseline. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Native deposited NovG regulatory-protein reference | is_gene_reference_for_expression_in | nov-VD2 background plus constitutive NovG plasmid pVD1 | NovG under ermE* in pUWL201; this does not ground the complete recombinant plasmid or assay sample. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| nov-VD2 background plus constitutive NovG plasmid pVD1 | restored_and_increased_production_of | Source-assigned novobiocin in later heterologous cultures | 59.8 mg/L, 299% of the intact empty-vector comparator. HPLC supernatant analysis at 305 nm against a standard; CDM flask cultures, 30 C, 200 rpm, seven days without antibiotics. Table III.1 means at least two independent mutants; row-specific n and uncertainty not given. Not the later deep-well qPCR baseline. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Native deposited NovG regulatory-protein reference | is_gene_reference_for_expression_in | nov-VD2 background plus native-promoter NovG plasmid pAE8 | pAE8 provides novG with its own promoter in the NovE-deficient host; no complete preparation identity claimed. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| nov-VD2 background plus native-promoter NovG plasmid pAE8 | restored_and_increased_production_of | Source-assigned novobiocin in later heterologous cultures | 34.6 mg/L, 173% of the intact comparator; expression can occur without NovE. HPLC supernatant analysis at 305 nm against a standard; CDM flask cultures, 30 C, 200 rpm, seven days without antibiotics. Table III.1 means at least two independent mutants; row-specific n and uncertainty not given. Not the later deep-well qPCR baseline. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Native deposited NovE regulatory-protein reference | is_gene_reference_for_expression_in | nov-AE10 NovG-deficient background plus pVD4 | pVD4 provides NovE in nov-AE10; not a direct NovE-NovG interaction or intact NovG replacement. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| nov-AE10 NovG-deficient background plus pVD4 | partially_restored_production_of | Source-assigned novobiocin in later heterologous cultures | 2.4 mg/L, 12%, versus 0.4 mg/L, 2%, for NovG-deficient empty-vector cells. Partial rescue is not absent rescue. The separate deltaG+pAE8 row reports 16 mg/L, 80%; keep this source percentage distinct from Eustaquio's 79%. HPLC supernatant analysis at 305 nm against a standard; CDM flask cultures, 30 C, 200 rpm, seven days without antibiotics. Table III.1 means at least two independent mutants; row-specific n and uncertainty not given. Not the later deep-well qPCR baseline. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 12 nodes, 12 edges
Condition-specific negative NovE EMSAs and detectable reciprocal regulator transcripts. No direct NovE-DNA event or NovE-NovG physical complex is asserted. Partial biosynthesis curation (review #589), not full-record sign-off. The cathomycin owner retains its unspecified sugar stereocentre; source-assigned novobiocin products remain ungrounded, not exact-key merged with CID 54675769. Native deposited sequences do not retrospectively identify every historical strain stock, complete preparation or engineered allele.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | encodes_native_reference | Native deposited NovE regulatory-protein reference | AF170880.4 CDS 4859..5512(+), 1-based inclusive, encodes AAF67498.1, 217 aa. CDS-aware table-11 translations across AF170880.2/.3/.4 exactly match the selected UniProt sequence. Old .2/.3 qualifiers are S. caeruleus, taxon 195949, NCIB 11891; .4 is S. niveus, taxon 193462, NCIMB 9219. The published correction assigns the deposit to 9219, not 11891. The thesis additionally prints 11897 in its strain table versus 11891 in results. These are retained source conflicts, not proof that all experimental stocks were identical. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novobiocin cluster underlying the cathomycin owner | encodes_native_reference | Native deposited NovG regulatory-protein reference | AF170880.4 CDS 6782..7738(+), 1-based inclusive, encodes AAF67500.1, 318 aa. CDS-aware table-11 translations across AF170880.2/.3/.4 exactly match the selected UniProt sequence. Old .2/.3 qualifiers are S. caeruleus, taxon 195949, NCIB 11891; .4 is S. niveus, taxon 193462, NCIMB 9219. The published correction assigns the deposit to 9219, not 11891. The thesis additionally prints 11897 in its strain table versus 11891 in results. These are retained source conflicts, not proof that all experimental stocks were identical. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Native deposited NovE regulatory-protein reference | is_native_sequence_reference_for | pAE18-derived N-terminal His6 NovE preparation | Native gene reference, not a complete tagged preparation or independently sequenced vector. NovE was expressed from pAE18/pRSET B in E. coli and purified by nickel affinity; the precise host stock and full fusion sequence remain unverified. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| pAE18-derived N-terminal His6 NovE preparation | was_tested_in | Later comparative NovE and NovG EMSAs | Approximately 4 ng DIG-end-labelled DNA; 3 ug NovE versus 1.5 ug NovG. Do not import the older 0.5 ug NovG assay's full buffer or kinetics; detailed methods are delegated to the 2008 paper, whose full text was not obtained. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Native deposited NovG regulatory-protein reference | is_native_sequence_reference_for | Later-study His6 NovG comparator preparation | Native gene reference, not a complete tagged preparation or independently sequenced vector. The later study reports a His6 NovG comparator; its complete sample identity was not independently verified or assumed identical to every earlier NovG preparation. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Later-study His6 NovG comparator preparation | was_tested_in | Later comparative NovE and NovG EMSAs | Approximately 4 ng DIG-end-labelled DNA; 3 ug NovE versus 1.5 ug NovG. Do not import the older 0.5 ug NovG assay's full buffer or kinetics; detailed methods are delegated to the 2008 paper, whose full text was not obtained. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Later comparative NovE and NovG EMSAs | gave_condition_specific_novE_result | No observed NovE shift in six tested novEFG-region fragments | Three upstream-of-novG fragments, one upstream-of-novF and two upstream-of-novE fragments showed no NovE shift. This does not prove universal inability to bind DNA. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1 |
| Later comparative NovE and NovG EMSAs | gave_novG_positive_comparator | Observed NovG shift at the novG-novH-region comparator | The source reproduced the known NovG fragment shift; this is not a protein-protein interaction or evidence that the differently loaded proteins have equal affinity. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Native deposited NovE regulatory-protein reference | is_coding_locus_reference_for_deletion_in | M512 nov-VD2 NovE-deficient RT-PCR condition | Gene-matched native reference, not the sequence of an intact protein in this deletion. 81 nt scar context, not native cassette replacement. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Native deposited NovG regulatory-protein reference | is_coding_locus_reference_for_deletion_in | M512 nov-AE10 NovG-deficient RT-PCR condition | Gene-matched native reference, not the sequence of an intact protein in this deletion. nov-AE10 context, not expression plasmid pAE12. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| M512 nov-VD2 NovE-deficient RT-PCR condition | retained_detectable_transcription | Detectable novG transcript without NovE | RT-PCR series at 22, 30, 44, 77, 94 h with hrdB control; DNase and no-RT controls in methods. Gel-detected transcript presence is not protein abundance or proof of unchanged expression. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1 |
| M512 nov-AE10 NovG-deficient RT-PCR condition | retained_detectable_transcription | Detectable novE transcript without NovG | Same RT-PCR series supports basal transcription in the reciprocal deletion. This rules out an absolute binary requirement under these conditions, not a quantitative regulatory effect. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 8 nodes, 8 edges
Later qPCR evidence supports a quantitative, condition-specific regulatory model, not a demonstrated direct NovE-NovG interaction. Partial biosynthesis curation (review #589), not full-record sign-off. The cathomycin owner retains its unspecified sugar stereocentre; source-assigned novobiocin products remain ungrounded, not exact-key merged with CID 54675769. Native deposited sequences do not retrospectively identify every historical strain stock, complete preparation or engineered allele.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited novobiocin cluster underlying the cathomycin owner | encodes_native_reference | Native deposited NovE regulatory-protein reference | AF170880.4 CDS 4859..5512(+), 1-based inclusive, encodes AAF67498.1, 217 aa. CDS-aware table-11 translations across AF170880.2/.3/.4 exactly match the selected UniProt sequence. Old .2/.3 qualifiers are S. caeruleus, taxon 195949, NCIB 11891; .4 is S. niveus, taxon 193462, NCIMB 9219. The published correction assigns the deposit to 9219, not 11891. The thesis additionally prints 11897 in its strain table versus 11891 in results. These are retained source conflicts, not proof that all experimental stocks were identical. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Deposited novobiocin cluster underlying the cathomycin owner | encodes_native_reference | Native deposited NovG regulatory-protein reference | AF170880.4 CDS 6782..7738(+), 1-based inclusive, encodes AAF67500.1, 318 aa. CDS-aware table-11 translations across AF170880.2/.3/.4 exactly match the selected UniProt sequence. Old .2/.3 qualifiers are S. caeruleus, taxon 195949, NCIB 11891; .4 is S. niveus, taxon 193462, NCIMB 9219. The published correction assigns the deposit to 9219, not 11891. The thesis additionally prints 11897 in its strain table versus 11891 in results. These are retained source conflicts, not proof that all experimental stocks were identical. | https://www.ncbi.nlm.nih.gov/nuccore/AF170880.4 |
| Native deposited NovE regulatory-protein reference | is_coding_locus_reference_for_deletion_in | M512 nov-VD2 48-hour qPCR condition | Gene-matched native reference, not the sequence of an intact protein in this deletion. Later qPCR context, not the earlier native experiment. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| Native deposited NovG regulatory-protein reference | is_coding_locus_reference_for_deletion_in | M512 NovG-deficient 48-hour qPCR condition | Gene-matched native reference, not the sequence of an intact protein in this deletion. Figure III.5 heading says nov-AE12, while caption/methods say nov-AE10. Retain this source conflict; pAE12 is separately a NovG expression plasmid. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| M512 nov-VD2 48-hour qPCR condition | showed_reduced_transcript_abundance | Reduced but nonzero normalized novG transcripts | novG signal was about 5% of the intact-cluster comparator, not zero. LightCycler SYBR qPCR with 10 ng RNA, normalized to hrdB, shown at 48 h. Deep-well culture context is distinct from the flask RT-PCR time course. Figure III.5 does not define replicate counts or uncertainty; no protein abundance inferred. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1
|
| M512 NovG-deficient 48-hour qPCR condition | showed_reduced_biosynthetic_transcription | Reduced novH, novO, novP, novQ transcript readouts | The measured biosynthetic loci here were novH, novO, novP and novQ, not every gene through novW. Strongly reduced readouts support NovG-dependent expression. The novH..novW operon is an inferred model, not directly sequenced full-length 18 kb RNA. Internal promoters remain condition-specific, not universally inactive. LightCycler SYBR qPCR with 10 ng RNA, normalized to hrdB, shown at 48 h. Deep-well culture context is distinct from the flask RT-PCR time course. Figure III.5 does not define replicate counts or uncertainty; no protein abundance inferred. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1 |
| Reduced but nonzero normalized novG transcripts | supports_quantitative_regulatory_model | Bounded NovE-NovG regulatory cascade model | Reduced abundance is compatible with the earlier detectable basal novG transcript. It supports a regulatory dependence without an absolute on/off gate or direct NovE binding mechanism. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1 |
| Reduced novH, novO, novP, novQ transcript readouts | supports_downstream_regulatory_model | Bounded NovE-NovG regulatory cascade model | NovG DNA-fragment binding is separate direct evidence. NovE phosphorylation, unknown partners or repression-release are proposed possibilities, not observed edges or new protein identities. Resistance-gene mechanism claims are not duplicated from AntibioticMech. | https://ub01.uni-tuebingen.de/xmlui/bitstream/handle/10900/49311/pdf/Dissertation_Dangel_090609_Printversion.pdf?isAllowed=y&sequence=1 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Explore across X-Mech
AntibioticMech (cathomycin) ↗
MediaIngredientMech (naturalproductmech:mibig-d4379ab45b) ↗
TaxonMech: Streptomyces niveus (NCBITaxon:193462) ↗
PathwayMech (mibig:BGC0000834) ↗
Provenance
| Source concept | Source | Version |
|---|---|---|
| BGC0000834 | MIBIG | 5 |
This page is generated from data/natural_products/shikimates_and_phenylpropanoids/cathomycin.yaml, which is generated from the committed inventories. Neither is edited by hand.