clorobiocin
naturalproductmech:mibig-619480541f MINTED SEEDED antibacterialenzyme inhibitor
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REVIEWED records curator sign-off on identity, structure, filing class and producer claims; DEPRECATED retains a superseded or out-of-scope record.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 | FJAQNRBDVKIIKK-LFLQOBSNSA-N |
|---|---|
| SMILES | CC1=CC=C(N1)C(=O)O[C@H]2[C@H]([C@@H](OC([C@@H]2OC)(C)C)OC3=C(C4=C(C=C3)C(=C(C(=O)O4)NC(=O)C5=CC(=C(C=C5)O)CC=C(C)C)O)Cl)O |
| Stereochemistry | fully defined |
| Cross-references | pubchem:54706138 |
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 roseochromogenus subsp. oscitans DS 12.976 NCBITaxon:1352936 |
BGC_CHARACTERIZED | mibig:BGC0000832 | PMID:12480894 |
Occurrences 1
Somebody found the compound in this organism and cited it. That is not a claim that the organism makes it, and nothing here promotes one to the other.
| Taxon | Source | Reference |
|---|---|---|
| Streptomyces NCBITaxon:1883 |
LOTUS | DOI:10.1002/CHIN.197249433 |
Biosynthetic gene clusters 1
| Accession | Host | Locus evidence | Genome |
|---|---|---|---|
| mibig:BGC0000832 | Streptomyces roseochromogenus subsp. oscitans DS 12.976 NCBITaxon:1352936 |
CLUSTER_DEMONSTRATED | genbank:AF329398.1 |
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.
Biosynthesis curated
Written by a curator with claim-level evidence on each step; not derived from a source. This is what the rest of the record exists to support.
| Step | Enzyme | Reaction | Substrate → product | Evidence |
|---|---|---|---|---|
| 10 | CloQ 4-hydroxyphenylpyruvate 3-dimethylallyltransferase UniProtKB:Q8GHB2 |
RHEA:37055 | CHEBI:36242 → CHEBI:74408 | DOI:10.1073/pnas.0337708100 |
Step 10: CloQ transfers the dimethylallyl group from dimethylallyl diphosphate to 3-(4-hydroxyphenyl)pyruvate(1-) to form the 3-dimethylallyl-4-hydroxyphenylpyruvate(1-) ring A precursor in the clorobiocin pathway.
Mechanism & Biosynthesis Overview
Causal graph — biosynthesis 3 nodes, 2 edges
Partial Streptomyces roseochromogenus clorobiocin biosynthesis graph for CloQ-catalyzed prenylation of the ring A precursor.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| 3-(4-hydroxyphenyl)pyruvate | is prenylated by | CloQ | DOI:10.1073/pnas.0337708100
|
|
| CloQ | forms | 3-dimethylallyl-4-hydroxyphenylpyruvate(1-) | This is a single curated ring A tailoring step; later assembly and tailoring steps to mature clorobiocin remain uncurated. | DOI:10.1073/pnas.0337708100
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 13 edges
Native CloL identity and recombinant ligation experiments from Galm 2003 (review #548). The assayed C-terminal His6 preparation is distinct from native Q8GHC3; literal primers predict a substitution and conflict with a printed coordinate and construct-map stop label. Experiments use novobiocin ring B, not the chlorinated clorobiocin acceptor. Formation of novobiocic acid and selected analogues is not synthesis of mature clorobiocin. Existing CloQ evidence is unchanged. Galm PDF page numbers exceed printed pages by four; Schmutz PDF numbers exceed them by ten.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native cloL locus | encodes_native_protein | Native CloL ligase | AF329398.1 cloL/AAN65228.1 occupies 18090..19673(+) and has a 1584-nt CDS including stop. CDS-aware table-11 translation yields 527 aa exactly matching deposited protein, Q8GHC3 and the complete printed Schmutz alignment. The native GTG initiates methionine. UniProt subspecies taxon 149682 and the locus strain taxon 1352936 (DS 12.976) have different taxonomic resolution, not conflicting protein identities. This accession does not denote the tagged preparation. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| pUG018-derived C-terminal His6 CloL preparation | was_constructed_from | Native cloL locus | The cloL/cloM subclone pCloLM, supplied by F. Pojer, provided the template for SphI/BglII cloning into pQE70, yielding reported 5.0-kb pUG018. Literal primers are cloL_P03f TCACGCATGCCGAACAAGGACCAC and cloL_P02r CTCAGATCTCCTGTCCACCAGCAC. The forward span is 18084..18107(+); the reverse exact 15-nt 3-prime match extrapolates to 19679..19656(-), versus the printed 19680..19657(-). A minimal 1596-nt literal-primer model predicts GTG-to-ATG start recoding, Ala2Pro in the native-length core, and TGA-to-AGA stop replacement. These are reconstructions, not sequenced experimental plasmid or fusion-protein properties. Methods remove the stop for the His6 fusion, but Figure 11 still labels TGA before His6; the conflict is retained. | 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 | pUG018-derived C-terminal His6 CloL preparation | The source describes 100 mL LB with 50 ug/mL carbenicillin, 30 C and 180 rpm, induction at OD600 0.7-0.8 with 0.5 mM IPTG, then three hours of expression. This is heterologous preparation of CloL, not native Streptomyces protein abundance or clorobiocin titer. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| pUG018-derived C-terminal His6 CloL preparation | has_purification_readout | CloL induction and Ni-NTA purification SDS-PAGE readout | Ni-NTA purification and Sephadex G-25 buffer exchange supplied the preparation. Figure 12 displays CloL lanes in order 2,3,4,1,5: before induction, after induction, soluble fraction, marker, and eluate. The source describes near homogeneity; no exact purity percentage is inferred. Protein was quantified by Bradford against BSA. NovL and CouL panels are separate preparations. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| pUG018-derived C-terminal His6 CloL preparation | catalysed_in | Recombinant CloL ligation assay with novobiocin ring B | The 100-uL assay supplied 1 mM aromatic acid, 1 mM novobiocin ring B, 5 mM ATP, 5 mM MnCl2, 50 mM Tris-HCl pH 8 and 0.5-2.0 ug protein for 30 min at 30 C; 5 uL 1.5 M TCA stopped the reaction. ATP is supplied here, not independently shown essential by these CloL measurements. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| 3-Dimethylallyl-4-hydroxybenzoic acid (DMAHB) | was_acyl_substrate_in | Recombinant CloL ligation assay with novobiocin ring B | DMAHB is the reference aromatic acid for the CloL assay and its normalization in the analogue panel. CouL instead uses its genuine pyrrole acid as the 100% reference. Its use is not a measurement of cellular precursor concentration. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| C-8-methylated novobiocin ring B | was_amine_substrate_in | Recombinant CloL ligation assay with novobiocin ring B | The acceptor is novobiocin ring B, methylated at C-8, not the chlorinated acceptor of mature clorobiocin. Assay activity is therefore not reported as a native clorobiocin-pathway turnover rate. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Heat-denatured CloL assay control | was_control_for | Recombinant CloL ligation assay with novobiocin ring B | The methods specify enzyme heated for 30 min at 100 C with the other assay constituents retained. This is the reported control design, not an invented quantitative zero-background trace. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Recombinant CloL ligation assay with novobiocin ring B | formed_assigned_product | Assay-assigned novobiocic acid | Products were extracted twice with 500 uL ethyl acetate; 900 uL pooled organic phase was dried at 30 C, dissolved in 105 uL methanol and 80 uL injected. HPLC detection was at 305 nm with novobiocic acid calibration, not the separate 340-nm clorobiocin culture assay. The assigned amide is novobiocic acid, not glycosylated mature clorobiocin. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Recombinant CloL ligation assay with novobiocin ring B | has_reported_specific_activity | CloL specific activity with DMAHB and novobiocin ring B | CloL is reported at 4.2 nkat/mg protein with DMAHB and novobiocin ring B. This is specific activity, not kcat, kcat/Km, or native-cell flux. The adjacent NovL 14.5 and CouL 7.4 nkat/mg values are not CloL measurements. Replicate counts and uncertainty definitions are not supplied for this value. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| pUG018-derived C-terminal His6 CloL preparation | was_tested_in | Relative aromatic-acid substrate panel | Figure 13 compares the reference acid, 22 synthetic analogues and the genuine CouL pyrrole acid with the same methylated ring-B acceptor. Each enzyme is normalized to its own genuine substrate at 100%: DMAHB for CloL/NovL, but 3-methylpyrrole-2,4-dicarboxylic acid for CouL. Bars are not absolute conversions or cross-enzyme activities. No exact bar heights, replicate count or error-bar definition are invented. Selected products have LC-MS/LC-MS-MS data in Table 20, not full NMR proof of every analogue. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Relative aromatic-acid substrate panel | showed_selected_acceptance | Selected acid analogues accepted under the tested conditions | The source reports CloL acceptance of 4-hydroxybenzoic acid (RAA500), the 3-chloro and 3-bromo derivatives RAA270/280, and side-chain amide analogues RAA240/250/260. These are selected qualitative positive observations under the panel conditions; they do not establish natural occurrence of the products. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Relative aromatic-acid substrate panel | showed_selected_nonacceptance | Selected acid analogues not accepted under the tested conditions | The genuine CouL 3-methylpyrrole-2,4-dicarboxylic acid (RAA400), 3-cyclohexyl-4-hydroxybenzoic acid (RAA520), and 3,5-dibromo-4-hydroxybenzoic acid (RAA290) were not accepted in this CloL panel. This is conditional nonacceptance, not universal inactivity, a measured binding constant, or an assigned quantitative detection threshold. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 13 nodes, 15 edges
Native CloHal and CloZ references, cassette-mutant observations and separate CloHal rescue in DS12.976. Genetic evidence is kept distinct from inferred enzyme mechanism; source length disagreements remain explicit. No exact substrate/order or physical assembly is asserted. Source-intake review #667; prior mechanisms and SEEDED status unchanged.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| CloHal native DS12.976 protein reference | is_native_reference_for_targeted_gene_product_in | Native-background clo-hal cassette mutants AE-h2, AE-h10 and AE-h11 | The native sequence reference is not the replacement allele. Three independently obtained mutants replace clo-hal with an apramycin-resistance cassette. FigureIII.3A/B and its caption show Southern genotyping. This is not the abandoned conventional in-frame strategy, nor a markerless deletion or full mutant-genome sequence. | https://www.uniprot.org/uniprotkb/Q8GHB4 |
| CloZ native DS12.976 uncharacterized protein reference | is_native_reference_for_targeted_gene_product_in | Native-background cloZ cassette mutants AE-Z4, AE-Z25 and AE-Z40 | Separate cassette-replacement experiment, FigureIII.3C/D. CloZ remains an uncharacterized PROTEIN, not a demonstrated enzyme. Native sequence grounding does not establish an intact historical preparation or universal pathway function. | https://www.uniprot.org/uniprotkb/Q8GHA5 |
| CloHal native DS12.976 protein reference | has_source_length_disagreement | Unresolved thesis/native protein-length discrepancies | Thesis PDF93 states525 amino acids; complete native CDS, GenPept and UniProt agree at524. No historical525-aa protein sequence or manuscript correction was established. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1 |
| CloZ native DS12.976 uncharacterized protein reference | has_source_length_disagreement | Unresolved thesis/native protein-length discrepancies | Thesis PDF93 states254 amino acids; complete native CDS, GenPept and UniProt agree at253. Preserve both source values without inventing a corrected or historical254-aa protein. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1 |
| Streptomyces roseochromogenus subsp. oscitans DS 12.976 | was_compared_in | Thesis production-culture extraction and HPLC comparison | The primary strain table identifies S. roseochromogenes var. oscitans DS12.976. Thesis production cultures and HPLC at340nm used authentic clorobiocin as comparison; these are distinct from the later2024 LC-MSE cultures. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Native-background clo-hal cassette mutants AE-h2, AE-h10 and AE-h11 | were_examined_by | Thesis production-culture extraction and HPLC comparison | All three independent mutants were cultured and extracted alongside the wild type. Independent mutant count is not a stated quantitative replicate count or detection limit. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Native-background clo-hal cassette mutants AE-h2, AE-h10 and AE-h11 | lost_detectable_production_of | Source-assigned clorobiocin | Reported clorobiocin production was abolished under the cited culture/extraction/HPLC conditions. No universal absence or quantitative zero is inferred. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Native-background clo-hal cassette mutants AE-h2, AE-h10 and AE-h11 | produced_structurally_assigned_congener | Source-assigned dechlorinated novclobiocin101 | The shorter-retention product was isolated and assigned using negative-ion FAB-MS and NMR; loss of the chlorine isotope pattern and nominal [M-H]-661 versus695 supported dechlorination. TableIII.1 explicitly marks overlapping or undeterminable signals. Novclobiocin101 is not equated to this owner's structure or assigned a new exact chemical identifier. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Native-background cloZ cassette mutants AE-Z4, AE-Z25 and AE-Z40 | retained_detectable_production_of | Source-assigned clorobiocin | All three cloZ mutants retained clorobiocin, identified by HPLC, negative-ion FAB-MS and proton NMR. The source describes a similar production level to wild type, not a statistical equivalence test. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Native-background cloZ cassette mutants AE-Z4, AE-Z25 and AE-Z40 | supports_condition_bounded_nonessentiality | CloZ nonessentiality limited to the tested culture conditions | Clorobiocin production persisted in these cassette mutants under the reported conditions. This does not establish universal dispensability, absence of biochemical activity, or a role under other conditions. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| CloHal native DS12.976 protein reference | is_native_reference_for_gene_expressed_in | AE-h10 carrying the clo-hal expression plasmid pAE-ha7 | The strain table names AE-h10/pAE-ha7. An intact clo-hal copy was expressed using pUWL201 with ermE* promoter; this is a distinct rescue organism, not the native protein itself. Full historical plasmid sequence was not obtained. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1 |
| AE-h10 carrying the clo-hal expression plasmid pAE-ha7 | restored_reported_production_of | Source-assigned clorobiocin | Reported HPLC chromatogram matched wild type and negative-ion FAB-MS confirmed the peak. No numerical rescue fraction or uncertainty is inferred. FigureIII.4D is the separate novO-engineered strain, not this CloHal rescue chromatogram. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Native-background clo-hal cassette mutants AE-h2, AE-h10 and AE-h11 | supports_genetic_involvement_in | Genetically supported involvement in clorobiocin chlorination | Loss of chlorinated product with dechlorinated-congener formation supports a chlorination role; it is not an isolated-enzyme substrate assay. The separate complementation result strengthens the gene-level assignment without fully excluding every cassette or expression-context effect. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| AE-h10 carrying the clo-hal expression plasmid pAE-ha7 | supports_genetic_involvement_in | Genetically supported involvement in clorobiocin chlorination | Restoration supports the CloHal assignment. It does not identify the exact physiological substrate, prove direct turnover of novclobiocin101, or establish a physical protein complex. | https://tobias-lib.uni-tuebingen.de/xmlui/bitstream/handle/10900/48693/pdf/diss_AE.pdf?isAllowed=y&sequence=1
|
| Genetically supported involvement in clorobiocin chlorination | has_unresolved_mechanism | Unresolved substrate, reaction order and biochemical mechanism | The thesis leaves halogenation timing and substrate open, listing carrier-bound and later intermediate candidates. Family-level flavin-reductase discussion is not purified CloHal reconstitution, a specific reductase partner, cofactor occupancy or measured kinetics. No pathway step number or exact substrate/product reaction is added. | 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 8 nodes, 8 edges
Culture-level halide observations remain separate from native genetic evidence and inferred CloHal properties. No purified-enzyme kinetics, exact reaction, congener identity or congener antimicrobial activity is adopted. Review #667.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| DS12.976 natural-producer culture experiment | was_tested_with | KBr-supplemented producer cultures | Separate2024 culture LC-MSE experiment, not thesis complementation. | DOI:10.1128/spectrum.00423-24
|
| KBr-supplemented producer cultures | was_associated_with | Changed chlorinated, brominated and nonhalogenated metabolite signals | Brominated and nonhalogenated signals increased while clorobiocin signals declined. Signal intensities are not absolute titers or enzyme rates. | DOI:10.1128/spectrum.00423-24
|
| DS12.976 natural-producer culture experiment | was_tested_with | Fluoride- or iodide-supplemented producer cultures | Culture supplementation, not purified CloHal substrate screening. | DOI:10.1128/spectrum.00423-24
|
| Fluoride- or iodide-supplemented producer cultures | had_reported_outcome | No detected fluorinated or iodinated clorobiocin variant | Condition-bounded nondetection; not universal impossibility of fluorination or iodination. | DOI:10.1128/spectrum.00423-24
|
| Changed chlorinated, brominated and nonhalogenated metabolite signals | supports_source_interpretation | Proposed CloHal halide-use interpretation | Authors suggest preferential bromide use with reduced turnover from culture profiles. Neither affinity nor turnover was measured as an enzyme kinetic constant. | DOI:10.1128/spectrum.00423-24
|
| Native CloHal reference; culture-based role inference | is_proposed_enzyme_in | Proposed CloHal halide-use interpretation | Native identity does not independently establish the assayed preparation, direct substrate or reaction order. | DOI:10.1128/spectrum.00423-24 |
| Proposed CloHal halide-use interpretation | has_evidence_limit | No purified kinetics or universally excluded halides | The2024 aglycone statement is not adopted as a demonstrated substrate. The older thesis explicitly leaves timing unresolved; culture data alone do not settle it. | DOI:10.1128/spectrum.00423-24 |
| No detected fluorinated or iodinated clorobiocin variant | has_evidence_limit | No purified kinetics or universally excluded halides | MITE0000419v1 labels model reactions as heterologous/in-vitro evidence. Those database labels are not adopted as primary assay proof; issue667 records the discrepancy. | https://github.com/mite-standard/mite_data/blob/6eacd913b2036376031072f69cbb33908f132998/mite_data/data/MITE0000419.json |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 20 nodes, 37 edges
Eleven CloQ engineered proteins from Metzger 2010, with native identity, literal-primer reconstructions, preparation measurements and limited folding controls (review #553). The native reaction is already curated and is not duplicated. Q8GHB2 denotes the native 324-aa protein, not a mutant, tag-bearing precursor or GSH-scar preparation. The thesis reports sequencing, but complete experimental plasmids were not independently obtained. All thesis page locators are PDF pages, twelve above printed pages.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native cloQ locus | encodes_native_protein | Native CloQ sequence reference | AF329398.1 cloQ/AAN65239.1 is CDS 31832..32806(+), 975 nt including stop. CDS-aware table-11 translation exactly matches the deposited protein and Q8GHB2, 324 aa. UniProt taxon 149682 is subspecies-level; the owner/BGC strain taxon 1352936 is DS 12.976, a resolution difference rather than a sequence mismatch. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| Native CloQ sequence reference | is_core_sequence_reference_for | pET28_CloQ-derived WT comparator | The native protein is distinct from the recombinant WT comparator. The crystallized WT deposit contains GSH before the 324-aa native core after thrombin cleavage; this is not a native N terminus. The precise experimental aliquot used for each kinetic determination was not independently sequenced. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| pET28_CloQ parental DNA construct | was_expression_construct_for | pET28_CloQ-derived WT comparator | The source uses the Keller pET28_CloQ construct, also written pET28a_CloQ in the plasmid table. His-tag removal is demonstrated for the crystallization preparation; neither construct spelling identifies a separate native protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| pET28_CloQ parental DNA construct | was_reported_template_for | CloQ site-directed construct preparation | QuikChange-style PCR followed by DpnI digestion, E. coli transformation and reported plasmid sequencing generated the panel. Most constructs use pET28_CloQ; pUM34 has conflicting pUM32/pUM33 template descriptions, retained on its own edge. Literal-primer models are reconstructed native cores, not verified complete experimental plasmids. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-C215S engineered protein (pUM28) | pUM28 is the reported C215S expression construct. Forward primer 5-prime ATGCCCGAGGACTACTCCGTCGCCATCACCCTC; reverse primer 5-prime GAGGGTGATGGCGACGGAGTAGTCCTCGGGCAT. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal native-CDS primer model gives the intended C215S change and otherwise preserves the native core. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-C215S engineered protein (pUM28) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 5.0 mg purified protein per L culture and final concentration 11.3 mg/mL for pUM28. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-C215A engineered protein (pUM29) | pUM29 is the reported C215A expression construct. Forward primer 5-prime ATGCCCGAGGACTACGCCGTCGCCATCACCCTC; reverse primer 5-prime GAGGGTGATGGCGACGGCGTAGTCCTCGGGCAT. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal native-CDS primer model gives the intended C215A change and otherwise preserves the native core. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-C215A engineered protein (pUM29) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 3.0 mg purified protein per L culture and final concentration 6.7 mg/mL for pUM29. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-E281G engineered protein (pUM32) | pUM32 is the reported E281G expression construct. Forward primer 5-prime GACTACGTCAAGGCCGGCCGCAGCTACACCGGC; reverse primer 5-prime GCCGGTGTAGCTGCGGCCGGCCTTGACGTAGTC. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal native-CDS primer model gives the intended E281G change and otherwise preserves the native core. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-E281G engineered protein (pUM32) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 3.7 mg purified protein per L culture and final concentration 12.2 mg/mL for pUM32. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-R160A engineered protein (pUM35) | pUM35 is the reported R160A expression construct. Forward primer 5-prime GGCCTGGCACACGTCGCGTTCGCAGCGGTGGATTGG; reverse primer 5-prime CCAATCCACCGCTGCGAACGCGACGTGTGCCAGGCC. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal native-CDS primer model gives the intended R160A change and otherwise preserves the native core. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-R160A engineered protein (pUM35) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 54 mg purified protein per L culture and final concentration 9.1 mg/mL for pUM35. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. This row used purchased competent cells, unlike the in-house competent cells used for the other variants; the larger recovery cannot be attributed to the substitution alone. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-R160Q engineered protein (pUM36) | pUM36 is the reported R160Q expression construct. Forward primer 5-prime GGCCTGGCACACGTCCAGTTCGCAGCGGTGGATTGG; reverse primer 5-prime CCAATCCACCGCTGCGAACTGGACGTGTGCCAGGCC. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal native-CDS primer model gives the intended R160Q change and otherwise preserves the native core. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-R160Q engineered protein (pUM36) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 7.0 mg purified protein per L culture and final concentration 3.5 mg/mL for pUM36. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-R66S engineered protein (pUM31) | pUM31 is the reported R66S expression construct. Forward primer 5-prime GATCAACTGTCGTACTCGTTCTTCTCCCGCCTC; reverse primer 5-prime GAGGCGGGAGAAGAACGAGTACGACAGTTGATC. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal native-CDS primer model gives the intended R66S change and otherwise preserves the native core. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-R66S engineered protein (pUM31) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 3.3 mg purified protein per L culture and final concentration 7.5 mg/mL for pUM31. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-K54S engineered protein (pUM37) | pUM37 is the reported K54S expression construct. Forward primer 5-prime GGCGCCACCTTATGGAGCACCACCAGTCAGCCTGG; reverse primer 5-prime CCAGGCTGACTGGTGGTGCTCCATAAGGTGGCGCC. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal native-CDS primer model gives the intended K54S change and otherwise preserves the native core. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-K54S engineered protein (pUM37) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 40 mg purified protein per L culture and final concentration 3.5 mg/mL for pUM37. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. This row used purchased competent cells, unlike the in-house competent cells used for the other variants; the larger recovery cannot be attributed to the substitution alone. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-E290stop engineered protein (pUM44) | pUM44 is the reported E290stop expression construct. Forward primer 5-prime CCGGCAACATGGCCTGAATCCTGGCCGGCTGG; reverse primer 5-prime CCAGCCGGCCAGGATTCAGGCCATGTTGCCGG. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal primer introduces a stop at native codon 290, predicting residues 1-289 and loss of 35 of the 324 native residues. Results prose instead reports removal of 37 residues. This conflict is not resolved by assuming the actual sample sequence equals the model; tag/scar residues are separate. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-E290stop engineered protein (pUM44) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 0.2 mg purified protein per L culture and final concentration 2.0 mg/mL for pUM44. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. Expression and purification were impaired. Additional anion-exchange chromatography was required and residual contamination was accepted; inactivity is not an isolated test of substrate access through the barrel end. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-F68S engineered protein (pUM30) | pUM30 is the reported F68S expression construct. Forward primer 5-prime CTGTCGTACCGGTTCTCCTCCCGCCTCAAGATG; reverse primer 5-prime CATCTTGAGGCGGGAGGAGAACCGGTACGACAG. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal native-CDS primer model gives the intended F68S change and otherwise preserves the native core. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-F68S engineered protein (pUM30) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 5.0 mg purified protein per L culture and final concentration 8.3 mg/mL for pUM30. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-R66S+F68S engineered protein (pUM33) | pUM33 is the reported R66S+F68S expression construct. Forward primer 5-prime GATCAACTGTCGTACTCGTTCTCCTCCCGCCTC; reverse primer 5-prime GAGGCGGGAGGAGAACGAGTACGACAGTTGATC. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The literal native-CDS primer model gives the intended R66S+F68S change and otherwise preserves the native core. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-R66S+F68S engineered protein (pUM33) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 4.5 mg purified protein per L culture and final concentration 7.2 mg/mL for pUM33. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ site-directed construct preparation | generated_expression_construct_for | CloQ-R66S+F68S+E281G engineered protein (pUM34) | pUM34 is the reported R66S+F68S+E281G expression construct. Forward primer 5-prime GATCAACTGTCGTACTCGTTCTCCTCCCGCCTC; reverse primer 5-prime GAGGCGGGAGGAGAACGAGTACGACAGTTGATC. The pair is exactly reverse-complementary and has a unique near-match at the expected native-CDS position. The plasmid table states pUM32 (E281G) with the R66S/F68S primers; results prose instead states pUM33. Applying these primers to the pUM32 native-core model yields R66S/F68S/E281G; applying them alone to the pUM33 double model does not introduce E281G. The source names a triple mutant, but its complete experimental construction remains unresolved. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-R66S+F68S+E281G engineered protein (pUM34) | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | Table 3.2 reports 10.0 mg purified protein per L culture and final concentration 10.7 mg/mL for pUM34. A280 quantification used 1.3 L/g/cm. These are recovered-protein measurements, not cellular expression rates or clorobiocin yields. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| pET28_CloQ-derived WT comparator | has_reported_preparation_in | CloQ expression, tag cleavage and purification workflow | WT crystallization preparation yielded 8.3 mg/L culture at 8.3 mg/mL. SDS-PAGE shows induction, soluble protein, tag cleavage and final material (Figure 3.10, including a 15-ug final lane). A280 coefficients were 1.2 L/g/cm with His tag and 1.3 without it. Trace impurities are described, not a quantified purity percentage. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| E. coli BL21(DE3)pLysS expression host | was_used_in | CloQ expression, tag cleavage and purification workflow | The 1-L TB culture was grown to OD600 0.6, cooled to 20 C, induced with 1 mM IPTG for the CloQ enzymes, and incubated six hours at 20 C/250 rpm. IMAC was followed by 0.1 U thrombin per mg protein during 15 h dialysis at 4 C, benzamidine/Ni-NTA removal of thrombin/tag, and Superdex 200. Final NaCl was adjusted to 50 mM and aliquots stored at -70 C. These are the crystallization/all-CloQ-mutant methods, not the separate tag-retaining NovL/CouL-chimera workflow. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ expression, tag cleavage and purification workflow | has_reported_solution_check | Reported solution homogeneity and monomer interpretation | The source interprets the WT and mutant preparations as homogeneous monomeric enzymes from dynamic light scattering, with E290stop explicitly exceptional for impaired preparation and contamination. Methods use filtered samples and at least 15 readings; full per-variant size distributions and independent biological replicates are not supplied here. This is not proof of native cellular stoichiometry, particularly not a purity certification of E290stop. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| pET28_CloQ-derived WT comparator | was_in_displayed_cd_comparison | Displayed CD comparison for WT and seven CloQ mutants | Figure 3.12 displays WT, R66S, F68S, C215S, C215A, E281G, R66S/F68S and R66S/F68S/E281G. Similar spectral shape supports a limited secondary-structure comparison, not identical folding at every site. R160A, R160Q, K54S and E290stop are not displayed. Methods specify 0.5 mg/mL protein in 20 mM phosphate pH 8 and a 190-260 nm scan; the caption says 250-190 nm. No unmeasured variant receives a CD folding control. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-C215S engineered protein (pUM28) | was_in_displayed_cd_comparison | Displayed CD comparison for WT and seven CloQ mutants | Figure 3.12 displays WT, R66S, F68S, C215S, C215A, E281G, R66S/F68S and R66S/F68S/E281G. Similar spectral shape supports a limited secondary-structure comparison, not identical folding at every site. R160A, R160Q, K54S and E290stop are not displayed. Methods specify 0.5 mg/mL protein in 20 mM phosphate pH 8 and a 190-260 nm scan; the caption says 250-190 nm. No unmeasured variant receives a CD folding control. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-C215A engineered protein (pUM29) | was_in_displayed_cd_comparison | Displayed CD comparison for WT and seven CloQ mutants | Figure 3.12 displays WT, R66S, F68S, C215S, C215A, E281G, R66S/F68S and R66S/F68S/E281G. Similar spectral shape supports a limited secondary-structure comparison, not identical folding at every site. R160A, R160Q, K54S and E290stop are not displayed. Methods specify 0.5 mg/mL protein in 20 mM phosphate pH 8 and a 190-260 nm scan; the caption says 250-190 nm. No unmeasured variant receives a CD folding control. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-E281G engineered protein (pUM32) | was_in_displayed_cd_comparison | Displayed CD comparison for WT and seven CloQ mutants | Figure 3.12 displays WT, R66S, F68S, C215S, C215A, E281G, R66S/F68S and R66S/F68S/E281G. Similar spectral shape supports a limited secondary-structure comparison, not identical folding at every site. R160A, R160Q, K54S and E290stop are not displayed. Methods specify 0.5 mg/mL protein in 20 mM phosphate pH 8 and a 190-260 nm scan; the caption says 250-190 nm. No unmeasured variant receives a CD folding control. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-R66S engineered protein (pUM31) | was_in_displayed_cd_comparison | Displayed CD comparison for WT and seven CloQ mutants | Figure 3.12 displays WT, R66S, F68S, C215S, C215A, E281G, R66S/F68S and R66S/F68S/E281G. Similar spectral shape supports a limited secondary-structure comparison, not identical folding at every site. R160A, R160Q, K54S and E290stop are not displayed. Methods specify 0.5 mg/mL protein in 20 mM phosphate pH 8 and a 190-260 nm scan; the caption says 250-190 nm. No unmeasured variant receives a CD folding control. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-F68S engineered protein (pUM30) | was_in_displayed_cd_comparison | Displayed CD comparison for WT and seven CloQ mutants | Figure 3.12 displays WT, R66S, F68S, C215S, C215A, E281G, R66S/F68S and R66S/F68S/E281G. Similar spectral shape supports a limited secondary-structure comparison, not identical folding at every site. R160A, R160Q, K54S and E290stop are not displayed. Methods specify 0.5 mg/mL protein in 20 mM phosphate pH 8 and a 190-260 nm scan; the caption says 250-190 nm. No unmeasured variant receives a CD folding control. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-R66S+F68S engineered protein (pUM33) | was_in_displayed_cd_comparison | Displayed CD comparison for WT and seven CloQ mutants | Figure 3.12 displays WT, R66S, F68S, C215S, C215A, E281G, R66S/F68S and R66S/F68S/E281G. Similar spectral shape supports a limited secondary-structure comparison, not identical folding at every site. R160A, R160Q, K54S and E290stop are not displayed. Methods specify 0.5 mg/mL protein in 20 mM phosphate pH 8 and a 190-260 nm scan; the caption says 250-190 nm. No unmeasured variant receives a CD folding control. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-R66S+F68S+E281G engineered protein (pUM34) | was_in_displayed_cd_comparison | Displayed CD comparison for WT and seven CloQ mutants | Figure 3.12 displays WT, R66S, F68S, C215S, C215A, E281G, R66S/F68S and R66S/F68S/E281G. Similar spectral shape supports a limited secondary-structure comparison, not identical folding at every site. R160A, R160Q, K54S and E290stop are not displayed. Methods specify 0.5 mg/mL protein in 20 mM phosphate pH 8 and a 190-260 nm scan; the caption says 250-190 nm. No unmeasured variant receives a CD folding control. | 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, 25 edges
CloQ WT and eleven mutant activity comparisons, with separate WT substrate-conditioned kinetic fits and endpoint product checks from Metzger 2010 (review #553). All measurements concern recombinant enzyme assays, not native clorobiocin production or each mutant binding a donor in a crystal. WT/variant preparations remain distinct from native Q8GHB2. PDF pages are twelve above printed pages.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| pET28_CloQ-derived WT comparator | was_reference_preparation_in | CloQ mutant PPi-coupled specific-activity panel | The coupled assay used 200 uL, 75 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 0.5 mM 4-HPP and 0.5 mM DMAPP or GPP, plus 66.7 uL PPi reagent prepared in 4 mL water. Following five minutes preincubation at 30 C, enzyme started a 15-minute NADH trace at 340 nm. Four coupling reactions oxidize two mol NADH per mol PPi, with extinction coefficient 6.22 mM^-1 cm^-1. The reported mutant loads vary from 5 to 50 ug according to activity; individual loads and an exact WT panel load are not assigned here. PPi-coupled rates are not direct structural identification of every product. MgCl2 was supplied despite CloQ being characterized as Mg-independent elsewhere. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-C215S engineered protein (pUM28) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | Source text reports 36% increased activity over WT with 4-HPP/DMAPP; the bar plot is consistent with retained or increased activity. GPP was not separately tested for this mutant in Figure 3.20. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-C215A engineered protein (pUM29) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | Source text reports 13% increased activity over WT with 4-HPP/DMAPP; the bar plot is consistent with retained or increased activity. GPP was not separately tested for this mutant in Figure 3.20. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-E281G engineered protein (pUM32) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | The source describes almost no activity with 4-HPP/DMAPP. The small plotted signal is not replaced by an exact zero. GPP was not separately tested for this mutant in Figure 3.20. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-R160A engineered protein (pUM35) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | The source reports approximately 20% residual WT activity with 4-HPP/DMAPP. GPP was not separately tested for this mutant in Figure 3.20. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-R160Q engineered protein (pUM36) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | The source reports approximately 20% residual WT activity with 4-HPP/DMAPP. GPP was not separately tested for this mutant in Figure 3.20. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-R66S engineered protein (pUM31) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | The source reports a 98% decrease relative to WT with 4-HPP/DMAPP. This is not a 98% loss of measured DMAPP binding affinity. GPP was not separately tested for this mutant in Figure 3.20. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-K54S engineered protein (pUM37) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | Source-described loss of activity applies to the supplied 4-HPP/DMAPP conditions. There is no stated quantitative detection threshold or displayed K54S CD spectrum. GPP was not separately tested for this mutant in Figure 3.20. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-E290stop engineered protein (pUM44) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | The truncated preparation is source-described as inactive with 4-HPP/DMAPP. Its recovery, purity, absent displayed CD and 35-versus-37-residue truncation discrepancy prevent attributing this solely to a barrel-access effect. GPP was not separately tested for this mutant in Figure 3.20. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-F68S engineered protein (pUM30) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | The approximately 50% WT activity statement refers to GPP turnover with 4-HPP, not DMAPP. Figure 3.20 also shows strongly reduced DMAPP activity, without an exact numerical bar table. Removing F68 did not improve GPP turnover in this experiment. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-R66S+F68S engineered protein (pUM33) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | The source describes the double mutant as inactive. Figure 3.20 includes both DMAPP and GPP conditions; inactivity is conditional, with no stated quantitative detection threshold. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-R66S+F68S+E281G engineered protein (pUM34) | has_reported_activity_in | CloQ mutant PPi-coupled specific-activity panel | The source describes the named triple mutant as inactive and Figure 3.20 includes DMAPP and GPP. The conflicting pUM34 template descriptions remain unresolved; actual full-sample sequence is not established by a literal-primer model. Figure 3.20 has no supplied error-bar definition or replicate count; no exact bar height, mutant Km/kcat, or in-vivo flux is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| CloQ-C215S engineered protein (pUM28) | supports_bounded_covalent_link_interpretation | Cys215 covalent tether is dispensable in the tested assay | Retained activity despite replacing the cysteine supports dispensability of the Cys215-substrate covalent tether in these in-vitro conditions. It does not measure the reversibility/rate of thiohemiketal formation in native cells or establish that the adduct never contributes physiologically. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-C215A engineered protein (pUM29) | supports_bounded_covalent_link_interpretation | Cys215 covalent tether is dispensable in the tested assay | Retained activity despite replacing the cysteine supports dispensability of the Cys215-substrate covalent tether in these in-vitro conditions. It does not measure the reversibility/rate of thiohemiketal formation in native cells or establish that the adduct never contributes physiologically. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| pET28_CloQ-derived WT comparator | was_measured_in | WT substrate-conditioned PPi-coupled kinetic fits | The same PPi/NADH method, with 2.5 mM MgCl2 at 30 C, was used for WT kinetic fits after checking enzyme-dose linearity. Aromatic-substrate series held DMAPP at 0.5 mM; donor series held 4-HPP at 0.5 mM. Table 3.4/Figure 3.21 give fitted Km, turnover and efficiencies, using GraphPad Prism 5.01. The printed +/- values lack a stated error definition in the inspected relevant methods/results, and no replicate count is assigned. Fits at a fixed cosubstrate concentration are not independent binding constants or kinetic measurements for every mutant. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| WT substrate-conditioned PPi-coupled kinetic fits | has_substrate_conditioned_fit_for | 4-HPP | For 4-HPP, reported Km is 16 +/- 3.9 uM, kcat 0.159 s^-1 and kcat/Km 10000 M^-1 s^-1. The fixed cosubstrate is DMAPP at 0.5 mM; enzyme load is 2 ug in 200 uL. Values are transcribed from Table 3.4, including its rounding; uncertainty type and independent replication are unspecified. They are WT-preparation values, not intrinsic constants assigned to every engineered protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| WT substrate-conditioned PPi-coupled kinetic fits | has_substrate_conditioned_fit_for | L-Tyrosine | For L-Tyrosine, reported Km is 680 +/- 129 uM, kcat 0.0083 s^-1 and kcat/Km 12 M^-1 s^-1. The fixed cosubstrate is DMAPP at 0.5 mM; enzyme load is 16 ug in 200 uL. Values are transcribed from Table 3.4, including its rounding; uncertainty type and independent replication are unspecified. They are WT-preparation values, not intrinsic constants assigned to every engineered protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| WT substrate-conditioned PPi-coupled kinetic fits | has_substrate_conditioned_fit_for | p-Coumaric acid | For p-Coumaric acid, reported Km is 874 +/- 227 uM, kcat 0.0138 s^-1 and kcat/Km 16 M^-1 s^-1. The fixed cosubstrate is DMAPP at 0.5 mM; enzyme load is 16 ug in 200 uL. Values are transcribed from Table 3.4, including its rounding; uncertainty type and independent replication are unspecified. They are WT-preparation values, not intrinsic constants assigned to every engineered protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| WT substrate-conditioned PPi-coupled kinetic fits | has_substrate_conditioned_fit_for | DMAPP | For DMAPP, reported Km is 25 +/- 4.0 uM, kcat 0.253 s^-1 and kcat/Km 10000 M^-1 s^-1. The fixed cosubstrate is 4-HPP at 0.5 mM; enzyme load is 2 ug in 200 uL. Values are transcribed from Table 3.4, including its rounding; uncertainty type and independent replication are unspecified. They are WT-preparation values, not intrinsic constants assigned to every engineered protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| WT substrate-conditioned PPi-coupled kinetic fits | has_substrate_conditioned_fit_for | GPP | For GPP, reported Km is 307 +/- 55 uM, kcat 0.0291 s^-1 and kcat/Km 95 M^-1 s^-1. The fixed cosubstrate is 4-HPP at 0.5 mM; enzyme load is 16 ug in 200 uL. Values are transcribed from Table 3.4, including its rounding; uncertainty type and independent replication are unspecified. They are WT-preparation values, not intrinsic constants assigned to every engineered protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| pET28_CloQ-derived WT comparator | was_tested_in | WT endpoint HPLC product check | Separate endpoint assays used 100 uL, 75 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 0.5 mM aromatic acceptor and 0.5 mM donor, 2 or 16 ug enzyme, ten minutes at 30 C, then 2 uL formic acid. Products were extracted with 500 uL ethyl acetate. HPLC monitored 275 nm for keto-4-HPP/tyrosine and 300 nm for enol-4-HPP/p-coumarate. Tautomerism complicated HPLC quantitation, motivating the separate coupled kinetic assay. Methods do not establish an identical product-validation panel for each mutant. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| WT endpoint HPLC product check | formed_source_assigned_product | Assay-assigned dimethylallyl-tyrosine | Dimethylallyl-tyrosine from L-tyrosine and DMAPP was additionally examined by LC-MS (performed by Stefanie Siebenberg). These source-assigned products are not mature clorobiocin, and LC-MS alone is not an independent full NMR/regiochemical/stereochemical reconstruction. No natural occurrence or new product record is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| WT endpoint HPLC product check | formed_source_assigned_product | Assay-assigned geranyl-4-HPP | Geranyl-4-HPP from 4-HPP and GPP was additionally examined by LC-MS (performed by Stefanie Siebenberg). These source-assigned products are not mature clorobiocin, and LC-MS alone is not an independent full NMR/regiochemical/stereochemical reconstruction. No natural occurrence or new product record is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| WT substrate-conditioned PPi-coupled kinetic fits | does_not_resolve | Unresolved physiological keto-versus-enol substrate choice | L-tyrosine and p-coumarate were used as analogues of keto- and enol-like acceptors. Their low but nonzero efficiencies do not identify the physiological 4-HPP tautomer used by CloQ. The source concludes this comparison did not establish a preference. Relative alternative-substrate efficiencies also do not measure intracellular substrate availability or flux. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Native CloQ sequence reference | is_native_reference_for | pET28_CloQ-derived WT comparator | UniProtKB:Q8GHB2 is the deposited native sequence reference for pET28_CloQ-derived WT 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/Q8GHB2 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 15 nodes, 18 edges
Four deposited CloQ structural states, exact scar/native sequence mappings, the Cys215 thiohemiketal and bounded mechanistic hypotheses (review #553). Direct coordinate observations, source interpretations and proposed donor contacts are separate. Detailed methods are from the inspected Metzger thesis; only the journal abstract was obtained. PDF pages are twelve above printed pages.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native CloQ sequence reference | is_core_reference_for | pET28_CloQ-derived WT comparator | All four deposited polymer sequences are 327 aa: GSH plus the native 324-aa Q8GHB2 sequence, except the intended C215S in 2XM5 and R66S in 2XM7. Author residue numbers equal native numbers; label_seq_id is author number plus three. The sequence-difference positions 218 and 69 are label numbering, not native C215S/R66S positions. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| pET28_CloQ-derived WT comparator | has_deposited_structural_state | PDB 2XLQ deposited CloQ structural state | 2XLQ: X-ray diffraction, 2.22 A; 316 modeled polymer residues versus 327 deposited. Revision history 2010-10-27 through 2024-11-13, version 1.5; coordinates inspected 2026-10-04. This WT-4HPP state came from preparation/crystallization with 2 mM 4-HPP in the buffers. Thesis Table 3.3 lists a 2.21-A high-resolution data limit, while the current deposited refinement resolution is 2.22 A. Modeled residues are native 1-313 plus GSH, not all 327 deposited residues. The I4122 crystal state is not a direct measurement of cellular flux or a solved donor-bound ternary complex. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| pET28_CloQ-derived WT comparator | has_deposited_structural_state | PDB 2XLY deposited CloQ structural state | 2XLY: X-ray diffraction, 3.10 A; 307 modeled polymer residues versus 327 deposited. Revision history 2010-10-27 through 2023-12-20, version 1.5; coordinates inspected 2026-10-04. The apo state was prepared and crystallized without 4-HPP. No non-polymer ligand atoms are deposited. Lower resolution limits fine comparison; the source notes a W122 side-chain displacement. The I4122 crystal state is not a direct measurement of cellular flux or a solved donor-bound ternary complex. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-C215S engineered protein (pUM28) | has_deposited_structural_state | PDB 2XM5 deposited CloQ structural state | 2XM5: X-ray diffraction, 1.85 A; 311 modeled polymer residues versus 327 deposited. Revision history 2010-10-27 through 2023-12-20, version 1.4; coordinates inspected 2026-10-04. The C215S crystals were soaked with 5 mM 4-HPP, but 4-HPP was not assigned in the density or deposited. EDO, formate and waters are present; these are not a bound prenyl donor. The I4122 crystal state is not a direct measurement of cellular flux or a solved donor-bound ternary complex. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ-R66S engineered protein (pUM31) | has_deposited_structural_state | PDB 2XM7 deposited CloQ structural state | 2XM7: X-ray diffraction, 2.22 A; 307 modeled polymer residues versus 327 deposited. Revision history 2010-10-27 through 2024-11-06, version 1.5; coordinates inspected 2026-10-04. The R66S crystals were soaked with 5 mM 4-HPP and contain the covalent acceptor adduct. EDO, formate and waters are also deposited. The source notes F68 moving into space left by the smaller Ser66 side chain. The I4122 crystal state is not a direct measurement of cellular flux or a solved donor-bound ternary complex. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| PDB 2XLQ deposited CloQ structural state | contains_reported_covalent_adduct | Crystal Cys215-linked 4-HPP thiohemiketal | The deposited covale record links CYS author215 SG to ligand 34H C2. Direct Euclidean coordinate distance is 1.883 A, matching the deposited link distance. All 13 ligand heavy atoms have modeled occupancy 1.00; that is a refinement assignment, not a solution binding fraction. The source interprets continuous density and tetrahedral geometry as a 4-HPP thiohemiketal. | https://files.rcsb.org/download/2XLQ.cif |
| PDB 2XLQ deposited CloQ structural state | has_observed_acceptor_contacts | Observed aromatic-acceptor contact environment | Using the CCD atom map, E281 OE1 to phenolic 34H O3 is 2.574 A; the shortest R160 guanidinium/carboxyl oxygen pair is NH2-O1 at 3.101 A. These are curator-calculated heavy-atom distances, not experimentally located hydrogens. The source interprets E281 hydrogen bonding and an R160 carboxyl interaction, with a surrounding hydrophobic environment including F68, W122, Y174, Y233 and L235. Contact geometry does not by itself establish proton-transfer direction, affinity or the essentiality of a residue. | https://files.rcsb.org/download/2XLQ.cif |
| PDB 2XM7 deposited CloQ structural state | contains_reported_covalent_adduct | Crystal Cys215-linked 4-HPP thiohemiketal | The deposited covale record links CYS author215 SG to ligand 34H C2. Direct Euclidean coordinate distance is 1.895 A, matching the deposited link distance. All 13 ligand heavy atoms have modeled occupancy 1.00; that is a refinement assignment, not a solution binding fraction. The source interprets continuous density and tetrahedral geometry as a 4-HPP thiohemiketal. | https://files.rcsb.org/download/2XM7.cif |
| PDB 2XM7 deposited CloQ structural state | has_observed_acceptor_contacts | Observed aromatic-acceptor contact environment | Using the CCD atom map, E281 OE1 to phenolic 34H O3 is 2.665 A; the shortest R160 guanidinium/carboxyl oxygen pair is NH2-O1 at 3.064 A. These are curator-calculated heavy-atom distances, not experimentally located hydrogens. The source interprets E281 hydrogen bonding and an R160 carboxyl interaction, with a surrounding hydrophobic environment including F68, W122, Y174, Y233 and L235. Contact geometry does not by itself establish proton-transfer direction, affinity or the essentiality of a residue. | https://files.rcsb.org/download/2XM7.cif |
| Crystal Cys215-linked 4-HPP thiohemiketal | has_covalent_context_in | Observed aromatic-acceptor contact environment | Standalone CCD 34H is named (2R)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid. Its bond table maps C2-O4 as the alpha hydroxyl and C7-O3 as the phenolic hydroxyl; a CCD covalent-modification annotation links C2 to CYS SG. The full crystal includes that protein-ligand covalent connection. The component name or isolated CCD formula is not evidence that CloQ makes free phenyllactate, and it is not equated with the unmodified CHEBI:36242 substrate. | https://files.rcsb.org/ligands/download/34H.cif |
| PDB 2XM5 deposited CloQ structural state | has_unassigned_density | Unassigned acceptor-region density in C215S | Despite a 5 mM 4-HPP soak, the source does not assign the acceptor-region density to 4-HPP or another ligand. Disorder/keto-enol exchange is suggested, not demonstrated. The observed Y233 displacement and R66 rotation do not locate the missing ligand or prove a different bound substrate pose. Absence of an assigned ligand is not absence of binding in solution. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| PDB 2XLQ deposited CloQ structural state | shows | CloQ PT-barrel architecture | Five repeated alpha-alpha-beta-beta units produce a ten-stranded antiparallel beta barrel surrounded by helices. The central cavity has polar and hydrophobic regions; the C-terminal region including helix 11 closes the lower end in the observed structure. This is not a TIM barrel or evidence that every substrate must use one experimentally tracked entry route. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| CloQ PT-barrel architecture | motivates_hypothesis | Hypothesized DMAPP coordination and cation shielding | No DMAPP, GPP or nonhydrolysable donor analogue was observed bound in these four deposits despite soaking/cocrystallization trials. The source schematic proposes K54, R66, K120, R229 and K279 as a positive diphosphate-binding region, with N172 and surrounding aromatic side chains contributing contacts/shielding. The putative K54 substitution for a metal charge and cation-pi shielding are hypotheses informed by homolog structures and mutagenesis, not observed CloQ donor contacts. Figure 3.19 is schematic, not a refined bound-donor model. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Observed aromatic-acceptor contact environment | motivates_hypothesis | Proposed dual catalytic-base roles of E281 | The source proposes E281 both activating the phenolic group and removing the proton from the aromatic sigma-complex. It explicitly treats the dual role as hypothetical because exact substrate positions are not secured. Severe E281G activity loss supports functional importance but does not independently prove either proton-transfer step or observe a carbocation intermediate. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| PDB 2XLQ deposited CloQ structural state | has_bounded_assembly_assignment | Condition-dependent oligomeric-state interpretation | 2XLQ assembly 1 is author-assigned monomeric; assembly 2 is a PISA-generated tetramer. Both assignments remain visible and are not collapsed into a proven biological tetramer. The thesis DLS interpretation concerns prepared protein in solution; crystallographic packing and software assemblies answer different questions. | https://files.rcsb.org/download/2XLQ.cif |
| PDB 2XLY deposited CloQ structural state | has_bounded_assembly_assignment | Condition-dependent oligomeric-state interpretation | 2XLY lists an author-and-software-assigned monomeric assembly. This assignment is not an independent physiological stoichiometry experiment. The thesis DLS interpretation concerns prepared protein in solution; crystallographic packing and software assemblies answer different questions. | https://files.rcsb.org/download/2XLY.cif |
| PDB 2XM5 deposited CloQ structural state | has_bounded_assembly_assignment | Condition-dependent oligomeric-state interpretation | 2XM5 lists an author-and-software-assigned monomeric assembly. This assignment is not an independent physiological stoichiometry experiment. The thesis DLS interpretation concerns prepared protein in solution; crystallographic packing and software assemblies answer different questions. | https://files.rcsb.org/download/2XM5.cif |
| PDB 2XM7 deposited CloQ structural state | has_bounded_assembly_assignment | Condition-dependent oligomeric-state interpretation | 2XM7 lists an author-and-software-assigned monomeric assembly. This assignment is not an independent physiological stoichiometry experiment. The thesis DLS interpretation concerns prepared protein in solution; crystallographic packing and software assemblies answer different questions. | https://files.rcsb.org/download/2XM7.cif |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 17 nodes, 20 edges
Native CloU/CloW references, heterologous DNA constructs and reconstructed junction/restriction limits; no purified CloU or CloW assay. Review #565. Host-conditioned genetic and source-assigned product evidence does not change the owner identity, source fields, previous graphs or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited clorobiocin cluster | contains | Native cloU locus | AF329398.1 CDS 36602..37864(+), AAN65243.1. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1
|
| Native cloU locus | encodes_native_reference | Native cloU sequence reference | CDS-aware table-11 translation gives 420 aa exactly matching AAN65243.1 and Q8GHA8. UniProt subspecies taxon149682 and deposited DS 12.976 strain taxon1352936 differ in taxonomic resolution, not native sequence. No engineered sample is assigned this accession. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| Deposited clorobiocin cluster | contains | Native cloW locus | AF329398.1 CDS 38863..39459(+), AAN65245.1. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1
|
| Native cloW locus | encodes_native_reference | Native cloW sequence reference | CDS-aware table-11 translation gives 198 aa exactly matching AAN65245.1 and Q8GHA6. UniProt subspecies taxon149682 and deposited DS 12.976 strain taxon1352936 differ in taxonomic resolution, not native sequence. No engineered sample is assigned this accession. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| Native cloU sequence reference | contains_proposed_motif | Proposed CloU SAM-associated sequence motif | Native residues108-116 are VVEFGSNTG, as printed in the thesis. The proposed SAM-binding association is sequence-based; no CloU-bound SAM structure, affinity or residue-mutagenesis assay is shown here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| Deposited clorobiocin cluster | was_carried_by | clo-BG1 intact-cluster cosmid | clo-BG1 carries the complete cluster and phiC31 integration functions. This is a DNA construct, not a protein complex. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-BG1 intact-cluster cosmid | was_replacement_template_for | clo-AF1 cassette-replacement cosmid | PCR-targeted lambda-RED replacement used the aac(3)IV cassette from pUG019. The source reports restriction screening, not independently supplied complete experimental plasmid sequences. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Native cloU locus | was_targeted_in | clo-AF1 cassette-replacement cosmid | Literal deletion primers: cloU-f GACCGGCGGTTTGCATCCCGTGAGGGGGAGGAAGAGTTGATTCCGGGGATCTCTAGATC; cloU-r TGGAACGGGATCACCGCAGCCGACACCGTGGCCACGTCAACTAGTCTGGAGCTGCTTC. Their unique39-nt native arms map36566..36604(+) and37900..37862(-), including the TTG start and TGA stop. These primers replace the intervening coding segment; the source shorthand entire ORF must not erase the retained ends. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| clo-AF1 cassette-replacement cosmid | was_excised_to_form | clo-AF2 cassette-excised cosmid | XbaI/SpeI digestion and ligation removed the cassette. Apramycin-sensitive, kanamycin-resistant clones were screened. Removal was intended to avoid polar effects; intent alone is not measured downstream expression. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF2 cassette-excised cosmid | has_reconstructed_junction | Literal-primer coding-remnant model | The literal-primer/XbaI-SpeI model retains TTGATTCCGGGGATCTCTAGTTGA:24 nt including start/stop, predicting MIPGISS with CDS-aware initiation and a net1239-nt deletion from the1263-nt native CDS. This is a reconstructed in-frame remnant, not a sequenced mutant or observed seven-residue protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| clo-BG1 intact-cluster cosmid | was_integrated_in | S. coelicolor M512 with integrated clo-BG1 | Intact-cluster heterologous comparator, not the native DS 12.976 producer. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF2 cassette-excised cosmid | was_integrated_in | S. coelicolor M512 with integrated clo-AF2 | PEG-mediated protoplast transformation and kanamycin selection generated phiC31-site integration strains. This is heterologous cluster deletion, not deletion in the native producer. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 with integrated clo-AF2 | had_reported_genotyping | Reported Southern genotyping | Five independent deletion isolates were reported to have the same genotype; FigureIII.6D displays one mutant lane against clo-BG1. The lost approximate1.0-kb band and4.1/3.9-kb signals are genotyping evidence, not an independently measured complete junction or copy number. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Reported Southern genotyping | has_restriction_label_conflict | BamHI versus BglII source-label conflict | Prose p.96 says BglII; FigureIII.6 caption defines B as BamHI and says genomic/cosmid DNA were BamHI-digested. The conflict is retained, not silently corrected. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Literal-primer coding-remnant model | provides_restriction_comparison_for | BamHI versus BglII source-label conflict | Native BglII cuts give 1077- and4115-bp adjacent fragments; the scar model joins them into3953bp. Native BamHI instead gives a5455-bp fragment spanning cloU. This supports the BglII interpretation of the local approximate1.0/4.1/3.9-kb sizes but does not prove the actual digest or reconstruct cosmid/host integration-junction bands. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| clo-BG1 intact-cluster cosmid | was_pcr_template_for | pAF16 cloU complementation construct | Literal k-cloU-f GACCGGCGGTTTGGATCCCGTGA; k-cloU-r TTCGGACCTTCTAGACACGTGG. The1354-nt PCR model has BamHI/XbaI top-strand cuts1327nt apart, consistent with the reported approximate1.3-kb insert. It was cloned through pBluescript SK(-) pAF15 into pUWL201 pAF16 under constitutive ermE. Primer-introduced changes lie outside the CDS; complete experimental plasmids were not obtained. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| pAF16 cloU complementation construct | was_expression_construct_for | pAF16-derived CloU expression product | The source names an intact cloU copy. No protein abundance, purified sample, tag sequence or complete experimental protein sequence is established by the rescue. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Native cloU sequence reference | is_native_core_reference_for | pAF16-derived CloU expression product | The complete native CDS including TTG initiation and TGA stop is unchanged in the literal rescue-amplicon model. That model is not direct resequencing of the experimental expressed protein. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| S. coelicolor M512 with integrated clo-AF2 | was_complemented_to_form | S. coelicolor M512 clo-AF2 plus pAF16 | The clo-AF2 strain received pAF16; the strain table reports kanamycin and thiostrepton resistance. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| pAF16 cloU complementation construct | was_introduced_into | S. coelicolor M512 clo-AF2 plus pAF16 | The rescue construct is separate from the integrated deletion cosmid and the native accession. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 15 nodes, 20 edges
Eight reported titers and bounded nondetection under a shared culture/HPLC protocol; deletion, intact-cluster and complemented hosts remain separate. Review #565. Host-conditioned genetic and source-assigned product evidence does not change the owner identity, source fields, previous graphs or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| S. coelicolor M512 clo-BG1 comparator | was_cultured_and_extracted_in | Culture-extract HPLC-UV production workflow | Source methods use a two-day YMG preculture at30 C/210rpm, two days in corn-starch medium at33 C/210rpm, then5mL into50mL distillers-solubles production medium in a500-mL flask for nine days at33 C/210rpm. The same protocol is specified for S. coelicolor cluster strains; these are not native-producer titers. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-BG1 comparator | had_reported_profile | Intact-cluster production profile | One intact-cluster comparator and five independent deletion isolates were reported as cultured. The printed titers lack clone-specific replicate values and uncertainty estimates; five genotyped isolates do not become five quantitative replicates for every value. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Culture-extract HPLC-UV production workflow | measured | Intact-cluster production profile | One-mL culture aliquots were acidified to pH4 and extracted twice with1mL ethyl acetate. Dried extracts were dissolved in200-500uL methanol;50-100uL was injected on RP18 at1mL/min with340-nm detection. Authentic clorobiocin was the stated standard. Distinct congener response factors and detection thresholds are not supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF2 deletion host | was_cultured_and_extracted_in | Culture-extract HPLC-UV production workflow | Source methods use a two-day YMG preculture at30 C/210rpm, two days in corn-starch medium at33 C/210rpm, then5mL into50mL distillers-solubles production medium in a500-mL flask for nine days at33 C/210rpm. The same protocol is specified for S. coelicolor cluster strains; these are not native-producer titers. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF2 deletion host | had_reported_profile | Deletion-host production profile | One intact-cluster comparator and five independent deletion isolates were reported as cultured. The printed titers lack clone-specific replicate values and uncertainty estimates; five genotyped isolates do not become five quantitative replicates for every value. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Culture-extract HPLC-UV production workflow | measured | Deletion-host production profile | One-mL culture aliquots were acidified to pH4 and extracted twice with1mL ethyl acetate. Dried extracts were dissolved in200-500uL methanol;50-100uL was injected on RP18 at1mL/min with340-nm detection. Authentic clorobiocin was the stated standard. Distinct congener response factors and detection thresholds are not supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF2 plus pAF16 | was_cultured_and_extracted_in | Culture-extract HPLC-UV production workflow | Source methods use a two-day YMG preculture at30 C/210rpm, two days in corn-starch medium at33 C/210rpm, then5mL into50mL distillers-solubles production medium in a500-mL flask for nine days at33 C/210rpm. The same protocol is specified for S. coelicolor cluster strains; these are not native-producer titers. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF2 plus pAF16 | had_reported_profile | Complemented-host production profile | One intact-cluster comparator and five independent deletion isolates were reported as cultured. The printed titers lack clone-specific replicate values and uncertainty estimates; five genotyped isolates do not become five quantitative replicates for every value. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Culture-extract HPLC-UV production workflow | measured | Complemented-host production profile | One-mL culture aliquots were acidified to pH4 and extracted twice with1mL ethyl acetate. Dried extracts were dissolved in200-500uL methanol;50-100uL was injected on RP18 at1mL/min with340-nm detection. Authentic clorobiocin was the stated standard. Distinct congener response factors and detection thresholds are not supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Intact-cluster production profile | has_reported_titer_for | Source-assigned clorobiocin | Reported 9.0 mg/L by culture-extract HPLC. This is a source production estimate, not purified enzyme activity, an error-bounded mean or evidence that this congener is the exact parent structure. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Intact-cluster production profile | has_reported_titer_for | Source-assigned isoclorobiocin | Reported 2.8 mg/L by culture-extract HPLC. This is a source production estimate, not purified enzyme activity, an error-bounded mean or evidence that this congener is the exact parent structure. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Intact-cluster production profile | has_reported_titer_for | Source-assigned novclobiocin101 | Reported 5.3 mg/L by culture-extract HPLC. This is a source production estimate, not purified enzyme activity, an error-bounded mean or evidence that this congener is the exact parent structure. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Deletion-host production profile | has_reported_titer_for | Source-assigned clorobiocic acid | Reported 20 mg/L by culture-extract HPLC. This is a source production estimate, not purified enzyme activity, an error-bounded mean or evidence that this congener is the exact parent structure. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Deletion-host production profile | has_reported_titer_for | Source-assigned novclobiocic acid106 | Reported 39 mg/L by culture-extract HPLC. This is a source production estimate, not purified enzyme activity, an error-bounded mean or evidence that this congener is the exact parent structure. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Deletion-host production profile | has_reported_titer_for | Source-assigned novclobiocin122 | Reported 1.5 mg/L by culture-extract HPLC. This is a source production estimate, not purified enzyme activity, an error-bounded mean or evidence that this congener is the exact parent structure. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Deletion-host production profile | has_reported_titer_for | Source-assigned novclobiocin123 | Reported 0.4 mg/L by culture-extract HPLC. This is a source production estimate, not purified enzyme activity, an error-bounded mean or evidence that this congener is the exact parent structure. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Deletion-host production profile | has_reported_nondetection_for | Source-assigned clorobiocin | Clorobiocin was not detected under the reported conditions; no numerical detection limit or universal zero-production claim is supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Complemented-host production profile | has_reported_titer_for | Source-assigned clorobiocin | Reported10 mg/L clorobiocin versus9.0 mg/L in the intact-cluster comparator. The source calls the amounts comparable; no statistical equivalence test or confidence interval is supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Complemented-host production profile | has_interpretation_limit | Host, quantitation and rescue interpretation limits | Genetic complementation supports a cloU-specific contribution. No direct cloV/cloW transcript or protein measurement is supplied; a rescue does not exclude every possible polar effect or establish isolated catalytic parameters. The displayed CloU rescue comparison has no separate empty-pUWL201 production panel. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Deletion-host production profile | has_interpretation_limit | Host, quantitation and rescue interpretation limits | Most reported mutant products are aglycones. Later pRHAM/pMPU sugar-supply engineering is a distinct experiment, not interchangeable with these baseline1.5/0.4-mg/L congeners or the pAF16 complementation measurement. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.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, 20 edges
Primary product structures, nominal ions, solvent conflicts and explicitly inferred CloU/CloW roles; no transfer of congener antibacterial assays. Review #565. Host-conditioned genetic and source-assigned product evidence does not change the owner identity, source fields, previous graphs or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| S. coelicolor M512 clo-AF2 deletion host | supplied_culture_for | Preparative isolation and structure assignment | A deletion host was grown in5L for preparative ethyl-acetate extraction and HPLC isolation. The general methods describe pH4 extraction, petroleum-ether pretreatment, Sephadex LH20 and preparative RP18 purification. Spectra concern isolated products, not direct purified CloU catalysis. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Preparative isolation and structure assignment | provided_congener_data_for | Study product-assignment evidence | The stated 5-L preparation targets novclobiocin 122 and 123. Aglycone assignments are also discussed and appendix spectra are supplied, but their exact aliquots are not identified as this same 5-L culture. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Study product-assignment evidence | documents_source_assigned_product | Source-assigned clorobiocic acid | Product assignment follows the thesis MS/NMR results and depicted structures. No new exact-structure record or parent-identity equivalence is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Study product-assignment evidence | documents_source_assigned_product | Source-assigned novclobiocic acid106 | Product assignment follows the thesis MS/NMR results and depicted structures. No new exact-structure record or parent-identity equivalence is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Study product-assignment evidence | documents_source_assigned_product | Source-assigned novclobiocin122 | Product assignment follows the thesis MS/NMR results and depicted structures. No new exact-structure record or parent-identity equivalence is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Study product-assignment evidence | documents_source_assigned_product | Source-assigned novclobiocin123 | Product assignment follows the thesis MS/NMR results and depicted structures. No new exact-structure record or parent-identity equivalence is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Source-assigned novclobiocin122 | has_reported_nmr_assignment | Novclobiocin122 sugar NMR assignment | In DMSO, TableIII.2 reports 4''-H3.44ppm triplet(J9.6Hz),5''-H3.71ppm dq(J6.4,9.2Hz), and a single 5-position methyl signal7''-H3 at1.18ppm doublet(J6.4Hz). The6'' methyl signal is absent. The source assigns an L-rhamnose-derived sugar with the axial5-methyl group replaced by H; this is the source structural interpretation, not an independently solved absolute structure. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Source-assigned novclobiocin123 | has_reported_nmr_assignment | Novclobiocin123 acyl-position NMR assignment | TableIII.2 gives2''-H5.36ppm triplet(overlap,Jundetermined),3''-H4.29ppm dd(J2.8,9.0Hz),4''-H3.38ppm triplet(J9.0Hz), and7''-H3 at1.19ppm doublet(J6.4Hz). The source places the5-methylpyrrole-2-carbonyl substituent at2''-O rather than3''-O. The two desmethyl products are positional congeners, not interchangeable parent identities. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Source-assigned novclobiocin122 | has_reported_ion_in | Negative FAB-MS product readouts | Results text assigns nominal m/z681 [M-H]- and a14-mass-unit decrease relative to clorobiocin. The negative FAB appendix labels681.0; this is not a high-resolution neutral exact mass. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Source-assigned novclobiocin123 | has_reported_ion_in | Negative FAB-MS product readouts | Results describe the same nominal negative ion as122; the appendix labels681.3. Preserve the displayed value without calling the two decimal values identical exact-mass measurements. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Source-assigned clorobiocic acid | has_reported_ion_in | Negative FAB-MS product readouts | Prose p.99 calls415 a molecular weight, whereas the negative FAB appendix labels414.1. Retain this distinction; do not silently turn either into a neutral monoisotopic mass. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Source-assigned novclobiocic acid106 | has_reported_ion_in | Negative FAB-MS product readouts | Prose reports380 and the negative FAB appendix labels379.9. The nonchlorinated aglycone assignment also uses an8'-H signal at6.66ppm; the source's stated35-Da comparison is not an independently reconciled exact-mass difference. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Source-assigned clorobiocic acid | has_nmr_source_discrepancy | Acid NMR solvent-label discrepancy | TableIII.2 and the appendix heading specify methanol/CD3OD for the acids, but the acquisition text on the appendix acid spectra reads DMSO. Methods also use the unusual word Dimethylsulfonamid, while TableIII.2 names Dimethylsulfoxid for122/123. No harmonized acid solvent is asserted. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Novclobiocin122 sugar NMR assignment | supports_genetic_role_assignment | Genetically supported axial5-C-methylation role | The source combines loss of the axial5-methyl group in the cloU mutant with complementation to assign a5-C-methylation role. It does not supply a purified CloU substrate panel, SAM stoichiometry or a kinetic constant. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| Native CloU sequence reference | has_genetically_supported_role | Genetically supported axial5-C-methylation role | This assignment concerns CloU in clorobiocin biosynthesis. Homology to NovU/CouU is not a substitute for experiments on those proteins. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| Novclobiocin122 sugar NMR assignment | motivates_pathway_inference | Source-inferred sugar3,5-epimerization | Source-assigned L-rhamnose-derived stereochemistry supports3,5-epimerization somewhere in the sugar pathway. End-product genetics is not direct observation of each nucleotide-sugar intermediate or a uniquely assigned epimerase reaction. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| Native CloW reference for the proposed epimerase | is_proposed_to_mediate | Source-inferred sugar3,5-epimerization | The thesis proposes CloW as the3,5-epimerase, informed by sequence similarity and the CloU-mutant products. No CloW deletion, purified CloW turnover or exclusion of heterologous host-enzyme contributions is shown in this experiment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| Source-inferred sugar3,5-epimerization | has_attribution_limit | Genetic inference versus purified catalysis boundary | Do not transfer this CloU-deletion interpretation into direct in-vivo NovW or CouW evidence. The separately curated NovW in-vitro isotope/kinetic study and this heterologous clorobiocin genetic experiment answer different questions. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Genetically supported axial5-C-methylation role | has_assay_limit | Genetic inference versus purified catalysis boundary | These data support a genetic biosynthetic role, not observed physical assembly, purified catalysis or proof of a unique native intermediate order. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Acid NMR solvent-label discrepancy | limits_comparison | Genetic inference versus purified catalysis boundary | Clorobiocin and the acids are listed in methanol whereas122/123 are listed in DMSO. Cross-solvent chemical shifts cannot be treated as isolated effects of methyl removal; the conflicting acid acquisition labels remain unresolved. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 18 nodes, 18 edges
Seven source-matched native references, qualified database roles, separate transformation branches and historical sequence limits. Review #566. Constructs, source-assigned congeners and inferred roles do not change the exact owner identity or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| S. coelicolor M512 clo-AF2 experimental parent | was_separately_transformed_with | pRHAM sugar-pathway construct | Separate experimental strains. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF2 experimental parent | was_separately_transformed_with | pMPU oleU construct | Separate experimental strains. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF2 experimental parent | was_separately_transformed_with | cos16F4 reporter construct | Separate experimental strains. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| CloM native reference | is_native_sequence_reference_for | S. coelicolor M512 clo-AF2 experimental parent | AF329398.1:19687..20859(+), AAN65229.1, 390 aa. CDS-aware translation, deposited protein and UniProt match exactly. This reference does not identify a resequenced experimental protein. Native DS 12.976; strain taxon1352936 versus subspecies taxon149682. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| CloS native reference | is_native_sequence_reference_for | S. coelicolor M512 clo-AF2 experimental parent | AF329398.1:34466..35332(+), AAN65241.1, 288 aa. CDS-aware translation, deposited protein and UniProt match exactly. This reference does not identify a resequenced experimental protein. Native DS 12.976; strain taxon1352936 versus subspecies taxon149682. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| OleL native reference | is_native_sequence_reference_for | pRHAM sugar-pathway construct | AF055579.2:3356..3970(+), AAD55452.1, 204 aa. CDS-aware translation, deposited protein and UniProt match exactly. This reference does not identify a resequenced experimental protein. Native S. antibioticus ATCC11891; AF055579.1 lacks this region. | https://www.ncbi.nlm.nih.gov/nuccore/AF055579.2 |
| OleS native reference | is_native_sequence_reference_for | pRHAM sugar-pathway construct | AF055579.2:3967..5037(+), AAD55453.1, 356 aa. CDS-aware translation, deposited protein and UniProt match exactly. This reference does not identify a resequenced experimental protein. Native S. antibioticus ATCC11891; AF055579.1 lacks this region. | https://www.ncbi.nlm.nih.gov/nuccore/AF055579.2 |
| OleE native reference | is_native_sequence_reference_for | pRHAM sugar-pathway construct | AF055579.2:5034..6041(+), AAD55454.1, 335 aa. CDS-aware translation, deposited protein and UniProt match exactly. This reference does not identify a resequenced experimental protein. Native S. antibioticus ATCC11891; AF055579.1 lacks this region. | https://www.ncbi.nlm.nih.gov/nuccore/AF055579.2 |
| OleU native reference | is_native_sequence_reference_for | pRHAM sugar-pathway construct | AF055579.2:6038..6925(+), AAD55455.1, 295 aa. CDS-aware translation, deposited protein and UniProt match exactly. This reference does not identify a resequenced experimental protein. Native S. antibioticus ATCC11891; AF055579.1 lacks this region. | https://www.ncbi.nlm.nih.gov/nuccore/AF055579.2 |
| ElmGT native reference | is_native_sequence_reference_for | cos16F4 reporter construct | AM900040.1:15166..16314(+), CAP12607.1, 382 aa. CDS-aware translation, deposited protein and UniProt match exactly. This reference does not identify a resequenced experimental protein. Tu2353 cos16F4 deposit; AJ300305.2/CAC16413.2 also matches. | https://www.ncbi.nlm.nih.gov/nuccore/AM900040.1 |
| OleU native reference | is_native_sequence_reference_for | pMPU oleU construct | pWHM3-based, ermE*-driven oleU; full experimental plasmid sequence unavailable. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| Co-carried oleNI DNA prefix | was_co_carried_in | pRHAM sugar-pathway construct | pEM4-based construct. Native SphI/XmnI top-strand cuts3288/7424 model4136nt: complete oleL/S/E/U and502nt of oleNI, not a full-length OleNI protein. Restriction geometry is a reconstruction, not plasmid resequencing. | https://www.caister.com/backlist/jmmb/v/v2/v2n3/04.pdf |
| Historical ElmGT CAC16413.1 translation | differs_from_current_reference | ElmGT native reference | AJ300305.1/CAC16413.1 translates to384aa, not current382aa; differences extend beyond terminal length. A revised deposit is not proof that experimental aliquots had different proteins. Do not assign Q9F2F9 to the historical sequence. | https://www.ncbi.nlm.nih.gov/nuccore/AJ300305.1 |
| OleL native reference | has_database_assigned_role | Proposed OleL nucleotide-sugar epimerization | UniProt proposes conversion of the D-configured 4-keto-2,6-dideoxysugar to its L-configured product; the reaction assignment is probable, not a purified pRHAM-protein measurement. | https://www.uniprot.org/uniprotkb/Q9RR30
|
| OleS native reference | has_database_assigned_role | Annotated dTDP-glucose synthesis | Formation from dTTP and glucose1-phosphate is annotated by similarity. Do not treat the pRHAM experiment as isolated OleS turnover. | https://www.uniprot.org/uniprotkb/Q9RR29
|
| OleE native reference | has_database_assigned_role | Annotated dTDP-glucose dehydration | Formation of dTDP-4-dehydro-6-deoxy-D-glucose is annotated by similarity; cofactor binding and individual catalytic rates were not measured in the pRHAM comparison. | https://www.uniprot.org/uniprotkb/Q9RR28
|
| OleU native reference | has_database_assigned_role | Proposed OleU nucleotide-sugar reduction | UniProt proposes reduction of the4-keto-2,6-dideoxy substrate to dTDP-L-olivose. This probable native reaction is distinct from the genetically supported rhamnose-supply use. | https://www.uniprot.org/uniprotkb/Q9RR27
|
| ElmGT native reference | has_database_assigned_role | Annotated ElmGT glycosyl transfer | UniProt assigns dTDP-L-rhamnose transfer to8-demethyltetracenomycin C. This produces the rhamnosyl intermediate, not directly the fully methylated elloramycin reporter. | https://www.uniprot.org/uniprotkb/Q9F2F9
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 19 edges
A distinct three-host engineering comparison; all twelve titers retain their experimental context. Review #566. Constructs, source-assigned congeners and inferred roles do not change the exact owner identity or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| clo-AF2 baseline host | was_compared_in | Culture-extract HPLC-UV comparison | S. coelicolor M512 background. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF2 plus pRHAM host | was_compared_in | Culture-extract HPLC-UV comparison | S. coelicolor M512 background. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF2 plus pMPU host | was_compared_in | Culture-extract HPLC-UV comparison | S. coelicolor M512 background. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Culture-extract HPLC-UV comparison | yielded_profile | FigureIII.10A profile | mg/L: novclobiocic acid106=49; clorobiocic acid=23; novclobiocin122=1.3; novclobiocin123=0.4. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Culture-extract HPLC-UV comparison | yielded_profile | FigureIII.10B profile | mg/L: novclobiocic acid106=18; clorobiocic acid=7.5; novclobiocin122=30; novclobiocin123=10. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Culture-extract HPLC-UV comparison | yielded_profile | FigureIII.10C profile | mg/L: novclobiocic acid106=25; clorobiocic acid=35; novclobiocin122=11; novclobiocin123=2. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF2 baseline host | has_own_control_profile | FigureIII.10A profile | Not the earlier CloU baseline. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF2 plus pRHAM host | has_own_product_profile | FigureIII.10B profile | Products122/123 assigned by MS/NMR. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF2 plus pMPU host | has_own_product_profile | FigureIII.10C profile | Not purified enzyme activity. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF2 plus pMPU host | carried_construct_encoding | pMPU-encoded OleU protein | Protein abundance was not measured. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| OleU native reference | is_native_reference_for | pMPU-encoded OleU protein | Canonical donor sequence, not a resequenced pMPU protein. | https://www.uniprot.org/uniprotkb/Q9RR27 |
| FigureIII.10C profile | motivates | Inferred CloS sugar-discrimination step | Genetic rescue supports a reduction bottleneck. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| CloS native reference | has_inferred_substrate_preference_in | Inferred CloS sugar-discrimination step | Proposed preference for 5-C-methylated sugar; no purified CloS kinetics. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| FigureIII.10B profile | motivates | Inferred CloM L-rhamnosyl transfer | Sugar supply permits glycoside formation. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| CloM native reference | has_inferred_donor_acceptance_in | Inferred CloM L-rhamnosyl transfer | Host transferases remain possible; NovM kinetics are not CloM measurements. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| Culture-extract HPLC-UV comparison | has_method_and_comparison_limits | Production-to-enzyme inference limits | Nine-day production:33C,210rpm; pH4 extraction, RP18/340nm, clorobiocin standard. No stated uncertainty or matched empty-vector panels. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Inferred CloS sugar-discrimination step | has_annotation_limit | Production-to-enzyme inference limits | Q8GHB0 automatically annotates unmethylated dTDP-L-rhamnose formation; that rule-based assignment is not a measured kinetic result. | https://www.uniprot.org/uniprotkb/Q8GHB0
|
| FigureIII.10B profile | has_fold_summary_limit | Production-to-enzyme inference limits | Reported8/26-fold summaries are not exact ratios of these rounded profiles. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Inferred CloM L-rhamnosyl transfer | has_attribution_limit | Production-to-enzyme inference limits | Specificity and expression effects are not independently separated. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 15 edges
Elloramycin serves as a heterologous pathway reporter, not as the clorobiocin owner identity. Review #566. Constructs, source-assigned congeners and inferred roles do not change the exact owner identity or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| ElmGT native reference | is_deposited_reference_for | cos16F4 reporter construct | Native cos16F4 reference, not an experimentally sequenced protein preparation. | https://www.ncbi.nlm.nih.gov/nuccore/AM900040.1 |
| cos16F4 reporter construct | was_introduced_into | M512 plus cos16F4 reporter control | Source reports no sugar-biosynthesis genes on cos16F4. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| M512 plus cos16F4 reporter control | was_analysed_in | Reporter HPLC and LC-MS workflow | TSB preculture; R5A,5days,30C,240rpm. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Reporter HPLC and LC-MS workflow | yielded_profile | FigureIII.9A reporter profile | Supernatant pH3 extraction; HPLC386nm and authentic-reference LC-MS. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| FigureIII.9A reporter profile | contained_assigned_product | Source-assigned 8-demethyltetracenomycin C | Conditional product assignment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| cos16F4 reporter construct | was_introduced_into | M512 clo-AF2 plus cos16F4 reporter host | Separate reporter host. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| M512 clo-AF2 plus cos16F4 reporter host | was_analysed_in | Reporter HPLC and LC-MS workflow | TSB preculture; R5A,5days,30C,240rpm. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Reporter HPLC and LC-MS workflow | yielded_profile | FigureIII.9B reporter profile | Supernatant pH3 extraction; HPLC386nm and authentic-reference LC-MS. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| FigureIII.9B reporter profile | contained_assigned_product | Source-assigned 8-demethyltetracenomycin C | Conditional product assignment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| FigureIII.9B reporter profile | contained_assigned_product | Source-assigned elloramycin | 19.7-min shoulder beside20.1-min peak; glycosylation plus cosmid-encoded methylation, not a quantified sugar pool. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| FigureIII.9B reporter profile | motivates | Inferred limited dTDP-L-rhamnose supply | Pathway-level inference. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| FigureIII.9A reporter profile | has_interpretation_limit | Reporter interpretation limits | No glycosides reported; differing plot scales preclude direct height comparisons. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Inferred limited dTDP-L-rhamnose supply | has_attribution_limit | Reporter interpretation limits | No intracellular concentration or unique enzyme attribution established. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| M512 plus cos16F4 reporter control | has_own_control_profile | FigureIII.9A reporter profile | FigureIII.9A is the M512 plus cos16F4 control. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| M512 clo-AF2 plus cos16F4 reporter host | has_own_product_profile | FigureIII.9B reporter profile | FigureIII.9B is the M512 clo-AF2 plus cos16F4 reporter host. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 10 nodes, 11 edges
Native identity, heterologous genetics and three published congener yields. Review #567. Exact owner identity and SEEDED status are unchanged.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native cloP locus | encodes_native_protein | CloP native reference | AF329398.1:29998..30831(+),AAN65238.1,277aa. Native DS12.976; subspecies taxon149682 versus strain1352936. Neither the engineered allele nor a purified preparation is assigned this accession. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| Native cloP locus | was_inactivated_in | cloP-inactivated cluster | 2005 deletion report; exact engineered boundaries unavailable. | DOI:10.1002/cbic.200500019
|
| cloP-inactivated cluster | was_expressed_in | S. coelicolor M512 engineered producer | Heterologous host, not the natural producer. | DOI:10.1002/cbic.200500019
|
| S. coelicolor M512 engineered producer | yielded_products_characterized_in | Deletion-study product characterization | 2005 MS and1H-NMR assignments; this is not a purified CloP activity assay. | DOI:10.1002/cbic.200500019
|
| Deletion-study product characterization | has_reported_product | Source-assigned novclobiocin112 | 2006 reported yield=13mg/L; lacks noviose4-O-methyl and carries MePC at3-OH. Source-assigned congener, not the clorobiocin owner. | DOI:10.1128/AAC.50.4.1136-1142.2006
|
| Deletion-study product characterization | has_reported_product | Source-assigned novclobiocin113 | 2006 reported yield=6mg/L; lacks noviose4-O-methyl and carries MePC at2-OH. Source-assigned congener, not the clorobiocin owner. | DOI:10.1128/AAC.50.4.1136-1142.2006
|
| Deletion-study product characterization | has_reported_product | Source-assigned novclobiocin120 | 2006 reported yield=4mg/L; lacks noviose4-O-methyl and carries MePC at4-OH. Source-assigned congener, not the clorobiocin owner. | DOI:10.1128/AAC.50.4.1136-1142.2006
|
| Deletion-study product characterization | supports_assignment_of | Genetically supported noviose4-O-methylation role | Genetic product pattern supports function, not catalytic constants. | DOI:10.1002/cbic.200500019
|
| CloP native reference | has_genetically_supported_role | Genetically supported noviose4-O-methylation role | Noviose O-methylation assignment; no isolated turnover inferred. | DOI:10.1002/cbic.200500019
|
| Deletion-study product characterization | has_method_provenance_limit | Deletion-study interpretation limits | 2006 delegates these production methods to earlier studies. Do not transfer its detailed novclobiocin103 extraction protocol or topoisomerase-assay replicates to these yields. No independent replication or uncertainty is established here. | DOI:10.1128/AAC.50.4.1136-1142.2006
|
| cloP-inactivated cluster | has_construct_evidence_limit | Deletion-study interpretation limits | Abstract-only access does not establish a deletion scar, primer sequence, rescue outcome, polar-effect exclusion or unique reaction order. | DOI:10.1002/cbic.200500019
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 9 nodes, 12 edges
Natural-producer product observations are distinct from CloP substrate hypotheses. Review #567. Exact owner identity and SEEDED status are unchanged.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| CloP native reference | is_native_sequence_reference_for | S. roseochromogenes DS12.976 natural producer | Deposited DS12.976 reference, not resequencing of the2024 culture. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| S. roseochromogenes DS12.976 natural producer | was_studied_by | Culture-supernatant LC-MSE and spectral annotation | DSMZ65 medium,7days,30C,180rpm; EtOAc extraction, negative-ion LC-MSE, GNPS/fragment annotation. Biological triplicates are reported for this study. | DOI:10.1128/spectrum.00423-24
|
| Culture-supernatant LC-MSE and spectral annotation | detected_source_assigned_product | Source-assigned compound667.2 | Reported[M-H]-667.2; lacks sugar O-methyl and pyrrole methyl. | DOI:10.1128/spectrum.00423-24
|
| Culture-supernatant LC-MSE and spectral annotation | detected_source_assigned_product | Source-assigned novclobiocin112 | Reported[M-H]-681.1; lacks sugar O-methyl. Distinct from novclobiocin109. | DOI:10.1128/spectrum.00423-24
|
| Source-assigned compound667.2 | supports_pathway_inference | Inferred acylation without prior sugar methylation | Assigned acylated, sugar-unmethylated product. | DOI:10.1128/spectrum.00423-24
|
| Source-assigned novclobiocin112 | supports_pathway_inference | Inferred acylation without prior sugar methylation | Assigned acylated, sugar-unmethylated product. | DOI:10.1128/spectrum.00423-24
|
| CloP native reference | has_untested_substrate_exclusion_hypothesis | Hypothesized CloP nonacceptance and shunt status | Proposed inability to methylate these acylated products; not measured inactivity. | DOI:10.1128/spectrum.00423-24
|
| Source-assigned compound667.2 | motivates | Hypothesized CloP nonacceptance and shunt status | Possible terminal shunt, not demonstrated metabolic fate. | DOI:10.1128/spectrum.00423-24
|
| Source-assigned novclobiocin112 | motivates | Hypothesized CloP nonacceptance and shunt status | Same hypothesis, not a substrate-challenge experiment. | DOI:10.1128/spectrum.00423-24
|
| Culture-supernatant LC-MSE and spectral annotation | has_structure_assignment_limit | Metabolomics-to-enzyme inference limits | Source-rounded mass labels and fragment-based assignments are retained. NMR of711B is not NMR validation of these compounds; no new chemical identifier or congener bioactivity is asserted. | DOI:10.1128/spectrum.00423-24
|
| Hypothesized CloP nonacceptance and shunt status | has_homolog_model_limit | Metabolomics-to-enzyme inference limits | NovP structure/docking informs the proposal, not an experimental CloP structure or kinetic test. | DOI:10.1128/spectrum.00423-24
|
| Hypothesized CloP nonacceptance and shunt status | has_unresolved_source_wording | Duplicated compound667 in source route wording | Discussion names667 in both possible methylation routes. This wording is not silently corrected into a demonstrated substrate-to-product edge. | DOI:10.1128/spectrum.00423-24
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 17 edges
Exact native references and bounded deletion construction. Review #568. Native references, constructs and source-assigned products remain distinct; owner identity and SEEDED status are unchanged.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited clorobiocin cluster | contains | Native cloN1 locus | AF329398.1:20853..21140(+), AAN65230.1. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1
|
| Native cloN1 locus | encodes_native_reference | CloN1 native reference | 95 aa; CDS-aware table11 translation exactly matches deposited protein and Q8GHC1. DS 12.976 strain taxon1352936 versus subspecies taxon149682. This is not a resequenced engineered sample. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| Deposited clorobiocin cluster | contains | Native cloN2 locus | AF329398.1:21150..22217(+), AAN65231.1. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1
|
| Native cloN2 locus | encodes_native_reference | CloN2 native reference | 355 aa; CDS-aware table11 translation exactly matches deposited protein and Q8GHC0. DS 12.976 strain taxon1352936 versus subspecies taxon149682. This is not a resequenced engineered sample. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| Deposited clorobiocin cluster | contains | Native cloN7 locus | AF329398.1:27026..27862(+), AAN65236.1. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1
|
| Native cloN7 locus | encodes_native_reference | CloN7 native reference | 278 aa; CDS-aware table11 translation exactly matches deposited protein and Q8GHB5. DS 12.976 strain taxon1352936 versus subspecies taxon149682. This is not a resequenced engineered sample. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1 |
| Deposited clorobiocin cluster | was_carried_by | clo-BG1 cluster cosmid | Intact cluster with phiC31 integration functions. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-BG1 cluster cosmid | was_targeting_template_for | clo-AF5 cloN1 replacement cosmid | aac(3)IV cassette from pUG019 replaces the cloN1 coding interior. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Native cloN1 locus | was_targeted_in | clo-AF5 cloN1 replacement cosmid | Table forward primer CACAGTGCAACACAATCGATGGGGGAATTACGCGAGATGATTCCGGGGATCTCTAGATC; methods instead CACAGTGGAACACAATCGATGGGGGAATTACGCGAGATGATTCCGGGGATCTCTAGATC. Reverse GGCCACGTATATACCTGATGTCCGCATCTGTTGCGCCTAACTAGTCTGGAGCTGCTTC. Method39-nt arms match20817..20855(+) and21176..21138(-); table forward has one upstream base mismatch. The actual experimental primer is not independently established. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| clo-AF5 cloN1 replacement cosmid | was_excised_to_form | clo-AF6 cassette-excised cloN1 cosmid | XbaI/SpeI digestion and compatible-end religation; apramycin-sensitive selection. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF6 cassette-excised cloN1 cosmid | has_reconstructed_junction | Reconstructed cloN1 coding remnant | ATGATTCCGGGGATCTCTAGTTAG is24nt including start/stop, with an18-nt internal scar; predicts MIPGISS. The native288-nt CDS is shortened by264nt. This is a model, not a sequenced mutant or observed peptide. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| clo-BG1 cluster cosmid | was_targeting_template_for | clo-AF3 cassette-retaining cloN7 cosmid | The tested cloN7 construct retains aac(3)IV. Listed clo-AF4 is not the plotted mutant. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Native cloN7 locus | was_targeted_in | clo-AF3 cassette-retaining cloN7 cosmid | Forward GGCAGACTCCCCAACAGCAGAGAGGACCAACTGAGCATGATTCCGGGGATCTCTAGATC; reverse AGTGTGCGTGGTGCGCCAGCACTCCGACAAGCACCGTTAACTAGTCTGGAGCTGCTTC. Unique39-nt arms match26990..27028(+) and27898..27860(-). TableII.15 instead prints26990..27027 and the reverse interval in ascending order. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| clo-AF3 cassette-retaining cloN7 cosmid | has_polar_effect_limit | Construct and polar-effect limits | Cassette retention relies on a presumed transcription-unit end, not measured absence of downstream effects. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF6 cassette-excised cloN1 cosmid | has_construct_limit | Construct and polar-effect limits | Cassette excision is not complete sequence verification or proof excluding every polar effect. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| CloN1 native reference | has_sequence_based_motif_interpretation | CloN1 motif interpretation and wording conflict | Native41-46=LGVDSL and44-46=Asp-Ser-Leu. Thesis prose says Asn-Ser-Leu but prints LGVDSL. Carrier-protein/4-prime-phosphopantetheine-site assignment is a sequence inference, not measured loading. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| CloN1 motif interpretation and wording conflict | has_interpretation_limit | Construct and polar-effect limits | No purified carrier loading, stable complex or catalytic rate follows from motif similarity. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 13 nodes, 23 edges
Separate mutant genotypes, product readouts and partial rescue. Review #568. Native references, constructs and source-assigned products remain distinct; owner identity and SEEDED status are unchanged.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| CloN1 native reference | is_native_reference_for_disrupted_locus_in | S. coelicolor M512 clo-AF6 mutant | clo-AF6, not a native producer or an expressed native-protein measurement. | https://www.uniprot.org/uniprotkb/Q8GHC1 |
| CloN7 native reference | is_native_reference_for_disrupted_locus_in | S. coelicolor M512 clo-AF3 mutant | clo-AF3, not the cassette-excised clo-AF4. | https://www.uniprot.org/uniprotkb/Q8GHB5 |
| S. coelicolor M512 clo-AF6 mutant | has_reported_genotyping_context | Genetics and product-assignment limits | Five integrants selected; FigureIII.1D shows two. EcoRI2.4/4.6-kb bands replaced by6.8kb. Selection count is not the number of quantitative titer replicates. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF3 mutant | has_reported_genotyping_context | Genetics and product-assignment limits | Ten integrants selected; FigureIII.2D shows one. BamHI7.2/2.4-kb bands replaced by9.8kb. No full integrated-genome sequence or quantitative replicate count is supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-BG1 comparator | was_studied_in | Culture-extract HPLC and product assignment | Nine-day production at33C/210rpm after YMG and corn-starch precultures; pH4 ethyl-acetate extraction. RP18 HPLC at340nm with clorobiocin reference; separate pyrrole analysis at262nm. No titer uncertainty or congener-specific response factors are supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF6 mutant | was_studied_in | Culture-extract HPLC and product assignment | Nine-day production at33C/210rpm after YMG and corn-starch precultures; pH4 ethyl-acetate extraction. RP18 HPLC at340nm with clorobiocin reference; separate pyrrole analysis at262nm. No titer uncertainty or congener-specific response factors are supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF3 mutant | was_studied_in | Culture-extract HPLC and product assignment | Nine-day production at33C/210rpm after YMG and corn-starch precultures; pH4 ethyl-acetate extraction. RP18 HPLC at340nm with clorobiocin reference; separate pyrrole analysis at262nm. No titer uncertainty or congener-specific response factors are supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-BG1 comparator | had_reported_main_product | Source-assigned clorobiocin | FigureIII.3A also identifies isoclorobiocin and novclobiocin101. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF6 mutant | had_reported_titer_for | Source-assigned novclobiocin104 | 35mg/L; FigureIII.3B. Congener lacking the acyl substituent, not the owner structure. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF3 mutant | had_reported_titer_for | Source-assigned novclobiocin104 | 10mg/L; FigureIII.3C. Different mutant, not a replicate of the35mg/L result. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF6 mutant | had_reported_product | Source-assigned novclobiocin107 | Additional18-min product, source assigned by FAB-MS/1H-NMR comparison. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF3 mutant | had_reported_minor_product | Source-assigned novclobiocin107 | Reported in much smaller amounts; no exact titer supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| Culture-extract HPLC and product assignment | has_product_structure_limit | Genetics and product-assignment limits | Negative FAB ions588 for104 and554 for107 differ by34; prose says35Da. TableIII.1 (250MHz,methanol) lacks pyrrole signals for both. No new exact chemical identifier is assigned. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF6 mutant | had_reported_nondetection_for | Source-assigned clorobiocin | Under the tested conditions; no quantitative detection threshold. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF3 mutant | had_reported_nondetection_for | Source-assigned clorobiocin | Under the tested conditions; not universal zero production. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF6 mutant | had_reported_product | Source-assigned free pyrrole-2-carboxylic acid | HPLC/authentic reference and1H-NMR comparison. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF3 mutant | had_reported_product | Source-assigned free pyrrole-2-carboxylic acid | Same study-level assignment; separate mutant culture. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 clo-AF6 mutant | was_complemented_to_form | clo-AF6 plus pAF30 complementation host | pAF30 carries cloN1 under constitutive ermE in pUWL201, via pGEM-T/pAF29. Literal primers ACGACACACTGGATCCCAATCGA and TCCCGGCCACGTCTAGACCTGATGTCCGCATCTGTTGCG model368nt and341nt between BamHI/XbaI cuts, preserving the native CDS. The39-nt reverse primer maps21180..21142, not printed21170..21149; plasmid-table coordinates also need caution. Complete experimental plasmid sequence was not obtained. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| clo-AF6 plus pAF30 complementation host | partially_restored_production_of | Source-assigned clorobiocin | Approximately60% of pre-inactivation productivity by HPLC, not complete rescue or a purified-enzyme rate. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-AF6 plus pAF30 complementation host | supports_assignment_of | Genetically supported pyrrolyl-transfer involvement | Partial genetic rescue supports cloN1 contribution but does not measure cloN2 expression or exclude every polar effect. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| CloN1 native reference | has_genetically_supported_involvement_in | Genetically supported pyrrolyl-transfer involvement | Loss-of-function/product evidence, not individually reconstituted transfer chemistry. | DOI:10.1002/cbic.200500252
|
| CloN7 native reference | has_genetically_supported_involvement_in | Genetically supported pyrrolyl-transfer involvement | CloN7 involvement in pyrrolyl attachment; no purified turnover inferred. | DOI:10.1002/cbic.200500252
|
| Genetically supported pyrrolyl-transfer involvement | has_interpretation_limit | Genetics and product-assignment limits | No inferred physical assembly, direct carrier-to-carrier transfer or congener antibacterial activity is added. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 15 nodes, 18 edges
Four recombinant constructs and unsuccessful soluble-enzyme preparation. Review #568. Native references, constructs and source-assigned products remain distinct; owner identity and SEEDED status are unchanged.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| E. coli XL1 Blue MRF- expression host | was_used_in | IPTG induction and SDS-PAGE comparison | Four constructs were tested; FigureIII.4 shows pAF21 at1.5-3.0mM IPTG, not all preparations. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-BG1 PCR template | was_pcr_template_for | pAF27 C-His6 CloN1 construct | pGEM-T/pAF26 then NcoI/BglII into pQE60. Literal310-nt amplicon retains the95-aa core but replaces stop by Arg. Forward20841..20864 and reverse21150..21124 differ from printed endpoints. Full fusion sequence is unverified. Primers GAATTACGCGCCATGGTAAGTGGG and CTGTTGCAGATCTGGCCTCGGCAGCGG. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| pAF27 C-His6 CloN1 construct | was_expression_construct_for | pAF27-derived CloN1 expression attempt | Separate engineered preparation, not the native UniProt reference. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| CloN1 native reference | is_native_reference_for | pAF27-derived CloN1 expression attempt | Reference only; tag and primer-dependent changes are not assigned the native accession. | https://www.uniprot.org/uniprotkb/Q8GHC1 |
| pAF27-derived CloN1 expression attempt | has_reported_outcome_in | IPTG induction and SDS-PAGE comparison | Expected10-kDa overexpression not detected; source says not shown. This does not establish native absence or inactivity. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-BG1 PCR template | was_pcr_template_for | pAF21 C-His6 CloN2 construct | pLitmus28/pAF17 then NcoI/BglII into pQE60. Literal1092-nt amplicon predicts Arg2Gly in the native-length core and stop replacement by Arg; not a sequenced experimental protein. Primers AGGCGCAACCCATGGGGACATCAGGT and CTGTGGGCTGAGATCTAAGTCCATCG. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| pAF21 C-His6 CloN2 construct | was_expression_construct_for | pAF21-derived insoluble CloN2 preparation | Separate engineered preparation, not the native UniProt reference. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| CloN2 native reference | is_native_reference_for | pAF21-derived insoluble CloN2 preparation | Reference only; tag and primer-dependent changes are not assigned the native accession. | https://www.uniprot.org/uniprotkb/Q8GHC0 |
| pAF21-derived insoluble CloN2 preparation | has_reported_outcome_in | IPTG induction and SDS-PAGE comparison | Source-assigned38.1-kDa band; insoluble under tested conditions and not cleanly purified. No activity assay established. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-BG1 PCR template | was_pcr_template_for | pAF20 N-His6 CloN2 construct | pLitmus28/pAF19 then BamHI/PstI into pRSETB. Literal1094-nt amplicon changes the native-start region; forward span21137..21163 differs from the printed start21136. Full vector-fusion sequence is unverified. Primers CATAGGCGCAACGGATCCGACATCAGG and CTGTGGGCTGCAGTCAAAGTCCATCG. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| pAF20 N-His6 CloN2 construct | was_expression_construct_for | pAF20-derived CloN2 expression attempt | Separate engineered preparation, not the native UniProt reference. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| CloN2 native reference | is_native_reference_for | pAF20-derived CloN2 expression attempt | Reference only; tag and primer-dependent changes are not assigned the native accession. | https://www.uniprot.org/uniprotkb/Q8GHC0 |
| pAF20-derived CloN2 expression attempt | has_reported_outcome_in | IPTG induction and SDS-PAGE comparison | CloN2 detected only with pAF21 in the reported comparison; no successful pAF20 purification is established. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| clo-BG1 PCR template | was_pcr_template_for | pAF23 N-His6 CloN7 construct | pLitmus28/pAF22 then PstI/HindIII into pRSETB. Literal861-nt amplicon alters the native-start region; forward span27014..27040 differs from printed27013..27040. The fusion is not unmodified native278-aa CloN7. Primers GGACCAACTGAGCCTGCAGATGACAAA and CGACAAGAAGCTTTATGCGGGACGTG. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1 |
| pAF23 N-His6 CloN7 construct | was_expression_construct_for | pAF23-derived CloN7 expression attempt | Separate engineered preparation, not the native UniProt reference. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| CloN7 native reference | is_native_reference_for | pAF23-derived CloN7 expression attempt | Reference only; tag and primer-dependent changes are not assigned the native accession. | https://www.uniprot.org/uniprotkb/Q8GHB5 |
| pAF23-derived CloN7 expression attempt | has_reported_outcome_in | IPTG induction and SDS-PAGE comparison | Expected34.4-kDa overexpression not detected; source says not shown. No purified CloN7 measurement follows. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.pdf?isAllowed=y&sequence=1
|
| IPTG induction and SDS-PAGE comparison | has_interpretation_limit | Expression and preparation limits | Changes to IPTG, temperature and other hosts did not improve the reported results. FigureIII.4 is a whole-expression comparison, not a purity, solubility or kinetic panel by itself. No native-protein abundance, universal inactivity or stable CloN complex is inferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/48957/pdf/Dissertation_Anja_Freitag.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
Native-producer genetics and later feeding challenge a historical pathway-order proposal. Review #568. Native references, constructs and source-assigned products remain distinct; owner identity and SEEDED status are unchanged.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| CloN2 native reference | is_native_reference_for_disrupted_locus_in | Native-producer cloN2 deletion mutant | DS12.976; in-frame deletion, not the M512 experiment. | https://www.uniprot.org/uniprotkb/Q8GHC0 |
| Native-producer cloN2 deletion mutant | accumulated_source_assigned_product | Source-assigned novclobiocin104 | NMR/LC-MS-supported assignment lacking the sugar acyl substituent. | DOI:10.1099/mic.0.26314-0
|
| Native-producer cloN2 deletion mutant | accumulated_source_assigned_product | Source-assigned free pyrrole-2-carboxylic acid | Free acid observed in culture extract. | DOI:10.1099/mic.0.26314-0
|
| Native-producer cloN2 deletion mutant | was_complemented_to_form | cloN2 plasmid-complemented native producer | Replicative plasmid carrying cloN2. | DOI:10.1099/mic.0.26314-0
|
| cloN2 plasmid-complemented native producer | restored_production_of | Source-assigned clorobiocin | Restoration reported; no quantitative degree invented. | DOI:10.1099/mic.0.26314-0
|
| Native-producer cloN2 deletion mutant | motivated_historical_interpretation | Conflicting historical methylation-order interpretations | 2003 proposed methylation after transfer; this is not established ordering. | DOI:10.1099/mic.0.26314-0
|
| CloN2 native reference | is_native_reference_for_targeted_locus_in | Later pyrrolyl-SNAC feeding study | Distinct later mutant-feeding study. | https://www.uniprot.org/uniprotkb/Q8GHC0 |
| CloN7 native reference | is_native_reference_for_targeted_locus_in | Later pyrrolyl-SNAC feeding study | No assumption of identical earlier constructs. | https://www.uniprot.org/uniprotkb/Q8GHB5 |
| Later pyrrolyl-SNAC feeding study | reported | Fed cloN1/cloN2 mutant observations | HPLC/HPLC-MS after pyrrolyl-2-carboxyl-N-acetylcysteamine thioester feeding. | DOI:10.1007/s00203-006-0190-9
|
| Fed cloN1/cloN2 mutant observations | includes_source_assigned_product | Source-assigned novclobiocin109 | Pyrrolyl transfer observed in cloN1 and cloN2 mutants. | DOI:10.1007/s00203-006-0190-9
|
| Later pyrrolyl-SNAC feeding study | reported | Fed cloN7 mutant observation | Transfer not observed in cloN7 mutant; no detection limit supplied. | DOI:10.1007/s00203-006-0190-9
|
| Source-assigned novclobiocin109 | challenged_historical_interpretation | Conflicting historical methylation-order interpretations | No further methylation to clorobiocin reported; contrasts with2003 proposal. | DOI:10.1007/s00203-006-0190-9
|
| Later pyrrolyl-SNAC feeding study | motivated | Proposed coordinated CloN6/CloN7 process | Additional mutant/acid observations support a coordinated-process proposal. | DOI:10.1007/s00203-006-0190-9
|
| Proposed coordinated CloN6/CloN7 process | has_assembly_evidence_limit | Abstract-access and mechanistic limits | Not direct physical-complex or stoichiometry evidence. | DOI:10.1007/s00203-006-0190-9
|
| Conflicting historical methylation-order interpretations | has_access_limit | Abstract-access and mechanistic limits | Uninspected doses, titers, construct endpoints and isolated-enzyme kinetics are not supplied. | DOI:10.1099/mic.0.26314-0 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 13 nodes, 13 edges
Four exact native references and bounded motif/database interpretation. Review #569. Native identity, experimental preparations, observations and inferred mechanisms remain distinct. This partial curation does not change compound identity, source-owned fields, earlier graphs or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Deposited clorobiocin cluster | contains | Native cloN3 locus | AF329398.1 CDS 22207..23337(+), AAN65232.1. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1
|
| Native cloN3 locus | encodes_native_reference | Native cloN3 protein reference | CDS-aware table-11 translation yields 376 aa exactly matching AAN65232.1 and Q8GHB9. UniProt subspecies taxon149682 and deposited DS12.976 strain taxon1352936 differ in resolution, not protein identity. No tagged, truncated or cassette-altered construct inherits this accession. | https://www.uniprot.org/uniprotkb/Q8GHB9 |
| Deposited clorobiocin cluster | contains | Native cloN4 locus | AF329398.1 CDS 23456..24961(+), AAN65233.1. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1
|
| Native cloN4 locus | encodes_native_reference | Native cloN4 protein reference | CDS-aware table-11 translation yields 501 aa exactly matching AAN65233.1 and Q8GHB8. UniProt subspecies taxon149682 and deposited DS12.976 strain taxon1352936 differ in resolution, not protein identity. No tagged, truncated or cassette-altered construct inherits this accession. | https://www.uniprot.org/uniprotkb/Q8GHB8 |
| Deposited clorobiocin cluster | contains | Native cloN5 locus | AF329398.1 CDS 24965..25234(+), AAN65234.1. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1
|
| Native cloN5 locus | encodes_native_reference | Native cloN5 protein reference | CDS-aware table-11 translation yields 89 aa exactly matching AAN65234.1 and Q8GHB7. UniProt subspecies taxon149682 and deposited DS12.976 strain taxon1352936 differ in resolution, not protein identity. No tagged, truncated or cassette-altered construct inherits this accession. | https://www.uniprot.org/uniprotkb/Q8GHB7 |
| Deposited clorobiocin cluster | contains | Native cloN6 locus | AF329398.1 CDS 25292..26977(+), AAN65235.1. | https://www.ncbi.nlm.nih.gov/nuccore/AF329398.1
|
| Native cloN6 locus | encodes_native_reference | Native cloN6 protein reference | CDS-aware table-11 translation yields 561 aa exactly matching AAN65235.1 and Q8GHB6. UniProt subspecies taxon149682 and deposited DS12.976 strain taxon1352936 differ in resolution, not protein identity. No tagged, truncated or cassette-altered construct inherits this accession. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Native cloN6 protein reference | has_sequence_motif | CloN6 CxxxCxxC motif at native residues 219-226 | Native CAHDCGWC occupies residues219-226 and matches CxxxCxxC. This sequence observation does not measure iron-sulfur occupancy or activity. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Native cloN6 protein reference | has_database_annotation | Automated CloN6 [4Fe-4S] cofactor annotation | UniProt cofactor statement is automated ECO:0000256/ARBA:ARBA00001966, not a measured CloN6 cofactor stoichiometry. | https://www.uniprot.org/uniprotkb/Q8GHB6
|
| Native cloN6 protein reference | has_source_discrepancy | Thesis native-length and homolog-label discrepancies | The thesis text and FigureIII.4a use560aa, whereas the deposit and current UniProt sequence have561aa. PDF p.82 prints94% identity to CouL while elsewhere discussing CouN6; no corrected homolog identity or94% comparison is asserted here. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| CloN6 CxxxCxxC motif at native residues 219-226 | motivated_sequence_based_proposal | Proposed radical-SAM and cobalamin-associated methylation | The source invokes an N-terminal cobalamin-binding region and radical-SAM-family motifs. Genetics and sequence similarity do not reconstitute the proposed chemistry. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Automated CloN6 [4Fe-4S] cofactor annotation | is_not_experimental_confirmation_of | Proposed radical-SAM and cobalamin-associated methylation | Automated cofactor assignment is not direct catalysis, B12 dependence or evidence for the proposed physiological substrate. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 13 edges
Primary-abstract evidence for activation, carrier loading and staged oxidation. Review #569. Native identity, experimental preparations, observations and inferred mechanisms remain distinct. This partial curation does not change compound identity, source-owned fields, earlier graphs or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native cloN3 protein reference | is_native_reference_for | Reported CloN3 flavoprotein preparation | The native reference is distinct from an experimental preparation whose tag, construct endpoints and purification are not supplied in the inspected abstract. | https://www.uniprot.org/uniprotkb/Q8GHB9 |
| Native cloN4 protein reference | is_native_reference_for | Reported experimental CloN4 adenylation enzyme | The native reference is distinct from an experimental preparation whose tag, construct endpoints and purification are not supplied in the inspected abstract. | https://www.uniprot.org/uniprotkb/Q8GHB8 |
| Native cloN5 protein reference | is_native_reference_for | Reported holo-CloN5 carrier preparation | The native reference is distinct from an experimental preparation whose tag, construct endpoints and purification are not supplied in the inspected abstract. | https://www.uniprot.org/uniprotkb/Q8GHB7 |
| L-proline substrate | was_activated_by | Reported experimental CloN4 adenylation enzyme | The abstract identifies CloN4 and CouN4 as adenylation enzymes selecting L-proline. | DOI:10.1021/bi0476329
|
| Reported experimental CloN4 adenylation enzyme | formed_activation_intermediate | L-prolyl-AMP intermediate | The reported activation intermediate is L-prolyl-AMP; no rate or assay composition invented. | DOI:10.1021/bi0476329
|
| L-prolyl-AMP intermediate | was_installed_on | Reported holo-CloN5 carrier preparation | The abstract reports subsequent thioester loading on holo-CloN5 and holo-CouN5. This does not establish all cross-pairings or an exact assay-specific carrier assignment. | DOI:10.1021/bi0476329
|
| Reported holo-CloN5 carrier preparation | provided_carrier_form_for | Reported prolyl-S-CloN5 carrier form | Phosphopantetheinyl thioester linkage, not free proline oxidation or mature clorobiocin production. | DOI:10.1021/bi0476329
|
| Reported prolyl-S-CloN5 carrier form | has_assay_assignment_limit | Carrier assignment and abstract-access limits | Reported CloN5 loading does not identify the exact carrier in the rapid-quench MS experiment. The assay substrate is therefore a separate node with unspecified CloN5/CouN5 identity. | DOI:10.1021/bi0476329
|
| Prolyl-S-PCP substrate of unspecified carrier identity | was_substrate_in | Rapid-quench ESI-FTMS CloN3 oxidation experiment | The rapid-quench MS experiment is described at PCP level; its exact CloN5/CouN5 carrier is not assigned from this abstract. | DOI:10.1021/bi0476329
|
| Reported CloN3 flavoprotein preparation | catalysed_oxidation_in | Rapid-quench ESI-FTMS CloN3 oxidation experiment | CloN3 is described as a flavoprotein dehydrogenase. No FAD occupancy, added-cofactor concentration or kinetic constant is inferred. | DOI:10.1021/bi0476329
|
| Rapid-quench ESI-FTMS CloN3 oxidation experiment | detected_transient_intermediate | Transient two-electron-oxidized pyrrolinyl-S-enzyme | Rapid quench and electrospray ionization-Fourier transform mass spectrometry detect a two-electron-oxidized intermediate before the fully oxidized product. | DOI:10.1021/bi0476329
|
| Rapid-quench ESI-FTMS CloN3 oxidation experiment | detected_oxidized_product | Four-electron-oxidized pyrrolyl-2-carboxy-S-PCP | The heteroaromatic pyrrole remains carrier-tethered. This is not methylation or transfer to clorobiocin sugar. | DOI:10.1021/bi0476329
|
| Rapid-quench ESI-FTMS CloN3 oxidation experiment | has_evidence_limit | Carrier assignment and abstract-access limits | Only the primary abstract was inspected. No unique MS carrier, all crossover combinations, tag sequences, time points, concentrations, enzyme kinetics or stoichiometric complex is asserted. | DOI:10.1021/bi0476329
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 17 edges
Eight expression constructs, their intermediates and bounded literal-primer models. Review #569. Native identity, experimental preparations, observations and inferred mechanisms remain distinct. This partial curation does not change compound identity, source-owned fields, earlier graphs or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native cloN6 protein reference | is_native_reference_for | clo-BG1 and D1A8 native-cluster PCR templates | Native CDS identity only; plasmid names identify experimental templates, not alternative UniProt entries. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| clo-BG1 and D1A8 native-cluster PCR templates | were_pcr_templates_for | pCA1, pCA3 and pCA9 PCR cloning intermediates | pCA1 uses D1A8 and f/r; pCA3 uses clo-BG1 and Q_f/Q_r; pCA9 uses clo-BG1 and the forward primer called QE_f in methods but Q_f in the table. pCA3 remained a pGEM-T intermediate: further pQE60 cloning was not performed. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA1, pCA3 and pCA9 PCR cloning intermediates | have_literal_primer_models | Literal-primer native-coordinate models | Literal primers: cloN6_f AATTGAACGCGGATCCGTTCCTGCTA; cloN6_r CCGTCTCCGAAGCTTCACGGGGTCA; cloN6Q_f AAATTGAACGGATCCATGTTCC; cloN6Q_r CTCCGTGAGATCTCGGGGTCA; cloN6G_r TCTCCGTGCGGCCGCGGGGTCAT; cloN6SQ_f AACGCGGGCATGCTCCTGCTA. Forward f model25278..25303 and reverse r26991..26967 differ from printed ends25304 and26992. Q_f models25277..25298; Q_r26987..26967, not the printed12-nt26976..26987 interval. G_r models26988..26966 versus printed end26989; SQ_f25283..25303. Some8-9nt suffixes are unique only within the source-named locus. Minimal amplicons: pCA1 1714nt, pCA3 1711nt, pCA4 1712nt, pCA5 1705nt, conditional pCA9 1715nt. At native coordinates pCA1 changes the initial ATG to a proline codon, pCA5 predicts F2L and stop-to-Arg, pCA3/pCA4 replace the stop with Arg, and conditional pCA9 preserves the native core. These are primer models, not complete fusion sequences or independent plasmid resequencing. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Literal-primer native-coordinate models | reveals_source_conflict | pCA5/1 and pCA6 restriction-fragment discrepancies | The SphI/BglII pCA5 model has cut positions13,1114 and1694: upstream SphI/SphI interval1101nt and downstream SphI/BglII interval580nt. Methods instead call these0.6kb and1.1kb respectively. The table assigns25283..26391 to C-terminal pCA5/1 and26391..26987 to N-terminal pCA6, conflicting with native orientation and the literal restriction model. These disagreements are retained rather than choosing an unverified corrected construct. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| pCA1, pCA3 and pCA9 PCR cloning intermediates | have_sequence_evidence_limit | Reported sequencing versus unavailable construct sequence files | The thesis reports PCR-product sequencing, but actual sequence files were not obtained. The Q_f/QE_f name ambiguity makes pCA9/pCA11 primer reconstruction conditional. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native cloN6 protein reference | is_native_reference_for_construct | pCA5 C-terminal His6 CloN6 construct | pQE70, T5 promoter; two-step SphI/BglII assembly via pCA5/1 and pCA6. The engineered protein is not assigned Q8GHB6. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Native cloN6 protein reference | is_native_reference_for_construct | pSH1 N-terminal His6 CloN6 construct | pRSETB, T7 promoter; BamHI/HindIII insert from pCA1. The engineered protein is not assigned Q8GHB6. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Native cloN6 protein reference | is_native_reference_for_construct | pCA7 N-terminal His6 CloN6 construct | pQE31, T5 promoter; BamHI/HindIII subclone of pSH1. The engineered protein is not assigned Q8GHB6. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Native cloN6 protein reference | is_native_reference_for_construct | pCA11 N-terminal His8 CloN6 construct | pHis8, T7 promoter; BamHI/HindIII insert from pCA9. The engineered protein is not assigned Q8GHB6. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Native cloN6 protein reference | is_native_reference_for_construct | pCA4 N-terminal GST CloN6 construct | pGEX-4T-1, Tac promoter; Q_f/G_r PCR of pCA3, BamHI/NotI cloning. The engineered protein is not assigned Q8GHB6. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Native cloN6 protein reference | is_native_reference_for_construct | Reported pET32a N-terminal Trx-His6 CloN6 construct | Construction relayed as personal communication from S. Keller and D. Lawson; no independently obtained construct sequence or unique plasmid name. The engineered protein is not assigned Q8GHB6. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Native cloN6 protein reference | is_native_reference_for_construct | pCA2 pGM9/pSH1 fusion construct | HindIII fusion of pSH1 with pGM9 for Streptomyces expression. The engineered protein is not assigned Q8GHB6. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Native cloN6 protein reference | is_native_reference_for_construct | pCA8 yeast CloN6 expression construct | BamHI/NotI fragment from pCA4 transferred into pYES2/NT-C. The engineered protein is not assigned Q8GHB6. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| pSH1 N-terminal His6 CloN6 construct | was_subcloned_into | pCA7 N-terminal His6 CloN6 construct | BamHI/HindIII fragment transfer. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pSH1 N-terminal His6 CloN6 construct | was_subcloned_into | pCA2 pGM9/pSH1 fusion construct | HindIII vector fusion. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA4 N-terminal GST CloN6 construct | was_subcloned_into | pCA8 yeast CloN6 expression construct | BamHI/NotI transfer into pYES2/NT-C. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA5/1 and pCA6 restriction-fragment discrepancies | qualifies_reconstruction_of | pCA5 C-terminal His6 CloN6 construct | Source assembly route retained with unresolved size/orientation labels. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 28 nodes, 35 edges
Host- and construct-specific expression outcomes, without invented purified catalysis. Review #569. Native identity, experimental preparations, observations and inferred mechanisms remain distinct. This partial curation does not change compound identity, source-owned fields, earlier graphs or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native cloN6 protein reference | is_native_reference_for | pCA5 C-terminal His6 CloN6 construct expression attempt | Native sequence reference for the intended engineered construct; this does not assert that full-length protein was obtained or assign the fusion a native accession. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| pCA5 C-terminal His6 CloN6 construct | was_used_for | pCA5 C-terminal His6 CloN6 construct expression attempt | pQE70, T5 promoter; two-step SphI/BglII assembly via pCA5/1 and pCA6. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA5 C-terminal His6 CloN6 construct expression attempt | had_reported_outcome | pca5 reported expression outcome | E. coli XL1Blue MRF' and BL21(DE3)/pLysS; no overexpression detected and purification unsuccessful (data not shown). | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pca5 reported expression outcome | has_interpretation_limit | Expression-outcome and activity limits | Gel size and failed expression do not measure purified activity, native abundance or universal inactivity. An unshown result is not a displayed quantitative assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native cloN6 protein reference | is_native_reference_for | pSH1 N-terminal His6 CloN6 construct expression attempt | Native sequence reference for the intended engineered construct; this does not assert that full-length protein was obtained or assign the fusion a native accession. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| pSH1 N-terminal His6 CloN6 construct | was_used_for | pSH1 N-terminal His6 CloN6 construct expression attempt | pRSETB, T7 promoter; BamHI/HindIII insert from pCA1. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pSH1 N-terminal His6 CloN6 construct expression attempt | had_reported_outcome | psh1 reported expression outcome | Both E. coli XL1Blue MRF' and BL21(DE3)/pLysS showed an approximately60-kDa band instead of expected67.9kDa. FigureIII.1 displays BL21(DE3)/pLysS with2mM IPTG. Purification unsuccessful. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| psh1 reported expression outcome | has_interpretation_limit | Expression-outcome and activity limits | Gel size and failed expression do not measure purified activity, native abundance or universal inactivity. An unshown result is not a displayed quantitative assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native cloN6 protein reference | is_native_reference_for | pCA7 N-terminal His6 CloN6 construct expression attempt | Native sequence reference for the intended engineered construct; this does not assert that full-length protein was obtained or assign the fusion a native accession. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| pCA7 N-terminal His6 CloN6 construct | was_used_for | pCA7 N-terminal His6 CloN6 construct expression attempt | pQE31, T5 promoter; BamHI/HindIII subclone of pSH1. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA7 N-terminal His6 CloN6 construct expression attempt | had_reported_outcome | pca7 reported expression outcome | Tested in XL1Blue MRF' and BL21(DE3)/pLysS; FigureIII.2 specifically shows XL1Blue MRF',1mM IPTG,37C, approximately60kDa instead of65.7kDa; purification unsuccessful in this LB experiment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pca7 reported expression outcome | has_interpretation_limit | Expression-outcome and activity limits | Gel size and failed expression do not measure purified activity, native abundance or universal inactivity. An unshown result is not a displayed quantitative assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native cloN6 protein reference | is_native_reference_for | pCA11 N-terminal His8 CloN6 construct expression attempt | Native sequence reference for the intended engineered construct; this does not assert that full-length protein was obtained or assign the fusion a native accession. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| pCA11 N-terminal His8 CloN6 construct | was_used_for | pCA11 N-terminal His8 CloN6 construct expression attempt | pHis8, T7 promoter; BamHI/HindIII insert from pCA9. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA11 N-terminal His8 CloN6 construct expression attempt | had_reported_outcome | pca11 reported expression outcome | E. coli BL21(DE3)/pLysS; FigureIII.2 at0.5mM IPTG,20C shows approximately60kDa instead of66.7kDa. Purification unsuccessful in this LB experiment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pca11 reported expression outcome | has_interpretation_limit | Expression-outcome and activity limits | Gel size and failed expression do not measure purified activity, native abundance or universal inactivity. An unshown result is not a displayed quantitative assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native cloN6 protein reference | is_native_reference_for | pCA4 N-terminal GST CloN6 construct expression attempt | Native sequence reference for the intended engineered construct; this does not assert that full-length protein was obtained or assign the fusion a native accession. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| pCA4 N-terminal GST CloN6 construct | was_used_for | pCA4 N-terminal GST CloN6 construct expression attempt | pGEX-4T-1, Tac promoter; Q_f/G_r PCR of pCA3, BamHI/NotI cloning. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA4 N-terminal GST CloN6 construct expression attempt | had_reported_outcome | pca4 reported expression outcome | E. coli BL21(DE3)/pLysS; FigureIII.5 uses1mM IPTG. Approximately70-kDa band instead of expected91.2kDa; purification unsuccessful. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pca4 reported expression outcome | has_interpretation_limit | Expression-outcome and activity limits | Gel size and failed expression do not measure purified activity, native abundance or universal inactivity. An unshown result is not a displayed quantitative assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native cloN6 protein reference | is_native_reference_for | Reported pET32a N-terminal Trx-His6 CloN6 construct expression attempt | Native sequence reference for the intended engineered construct; this does not assert that full-length protein was obtained or assign the fusion a native accession. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| Reported pET32a N-terminal Trx-His6 CloN6 construct | was_used_for | Reported pET32a N-terminal Trx-His6 CloN6 construct expression attempt | Construction relayed as personal communication from S. Keller and D. Lawson; no independently obtained construct sequence or unique plasmid name. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Reported pET32a N-terminal Trx-His6 CloN6 construct expression attempt | had_reported_outcome | trx reported expression outcome | E. coli BL21(DE3); shortened product reported instead of full-length CloN6, data not shown. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| trx reported expression outcome | has_interpretation_limit | Expression-outcome and activity limits | Gel size and failed expression do not measure purified activity, native abundance or universal inactivity. An unshown result is not a displayed quantitative assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native cloN6 protein reference | is_native_reference_for | pCA2 pGM9/pSH1 fusion construct expression attempt | Native sequence reference for the intended engineered construct; this does not assert that full-length protein was obtained or assign the fusion a native accession. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| pCA2 pGM9/pSH1 fusion construct | was_used_for | pCA2 pGM9/pSH1 fusion construct expression attempt | HindIII fusion of pSH1 with pGM9 for Streptomyces expression. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA2 pGM9/pSH1 fusion construct expression attempt | had_reported_outcome | pca2 reported expression outcome | S. lividans T7; no overexpression under tested conditions, data not shown. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pca2 reported expression outcome | has_interpretation_limit | Expression-outcome and activity limits | Gel size and failed expression do not measure purified activity, native abundance or universal inactivity. An unshown result is not a displayed quantitative assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native cloN6 protein reference | is_native_reference_for | pCA8 yeast CloN6 expression construct expression attempt | Native sequence reference for the intended engineered construct; this does not assert that full-length protein was obtained or assign the fusion a native accession. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| pCA8 yeast CloN6 expression construct | was_used_for | pCA8 yeast CloN6 expression construct expression attempt | BamHI/NotI fragment from pCA4 transferred into pYES2/NT-C. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA8 yeast CloN6 expression construct expression attempt | had_reported_outcome | pca8 reported expression outcome | S. cerevisiae INVSc1 in the methods; expected71.8-kDa protein not overexpressed, and no other induced protein reported; data not shown. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pca8 reported expression outcome | has_interpretation_limit | Expression-outcome and activity limits | Gel size and failed expression do not measure purified activity, native abundance or universal inactivity. An unshown result is not a displayed quantitative assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Shared expression-method ranges | describes_methods_for | Native cloN6 protein reference | Shared E. coli methods use100mL LB,OD600 0.5-0.7,16/30/37C,170rpm, IPTG0.1/0.5/1/1.5/2mM and6or20h. This is not a fully crossed matrix for every construct; figure-specific20C conditions are reported separately. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pSH1 N-terminal His6 CloN6 construct expression attempt | was_tested_in | pSH1 cobalt, trace-element and methylcobalamin trials | BL21(DE3)/pLysS in LB with1uM cobalt chloride and/or1uM methylcobalamin and/or100uL trace-element solution per100mL. Source reports persistence of the approximately60-kDa product, not restoration of expected67.9-kDa protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pSH1 cobalt, trace-element and methylcobalamin trials | has_interpretation_limit | Expression-outcome and activity limits | Unsuccessful supplementation does not establish cofactor independence or identify a degradation mechanism. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 24 nodes, 24 edges
Ten sequence-verified peptides, helper expression and a separate unconfirmed purification band. Review #569. Native identity, experimental preparations, observations and inferred mechanisms remain distinct. This partial curation does not change compound identity, source-owned fields, earlier graphs or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native cloN6 protein reference | is_native_reference_for | pCA7 expression attempt in E. coli XL1Blue MRF' | N-His6 engineered construct, not an unmodified native protein. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| pCA7 expression attempt in E. coli XL1Blue MRF' | was_tested_with | pACYCisc coexpression trial | pACYCisc diagram includes fdx,hscA,hscB,iscA,iscU,iscS; FigureIII.3b at0.1and1mM IPTG,30C in E. coli XL1Blue. No uniquely verified accession is selected for these helper preparations. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pACYCisc coexpression trial | retained_expression_outcome | Approximately60-kDa band in the pACYCisc coexpression panel | Approximately60kDa remained; no expected65.7-kDa overexpression, and purification unsuccessful. This panel is not identified as the source of the excised MS aliquot. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA7 expression attempt in E. coli XL1Blue MRF' | yielded_gel_band | Approximately60-kDa pCA7 gel band excised for MS | The band examined by MS came from the pCA7 E. coli XL1Blue MRF' expression work. The source does not establish that the excised aliquot was from the pACYCisc panel. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Approximately60-kDa pCA7 gel band excised for MS | was_examined_by | Gel-excised band trypsin LC-MS analysis | Band excision from SDS gel, trypsin digestion and LC-MS; not an activity assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Gel-excised band trypsin LC-MS analysis | supported_sequence_identification | CloN6 peptide coverage with unresolved proteoform boundaries | Ten detected peptides map exactly to native Q8GHB6 and span68-555. Coverage is discontinuous and is not the complete proteoform sequence. The source calculates56.5kDa for68-555 and compares approximately60kDa on gel with65.7kDa for tagged full protein; it explicitly cannot locate the cleavage boundary. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | did_not_detect | Undetected N-terminal tryptic peptides | Targeted spectral searches did not find native-position peptides 1-13 MFLLHAPSVYDFR; 16-46 DDILFAYLSDSDSVNVTSVYEMYPIGWFSIK; 47-67 QQLADHDFDCQIVNIASLMLK. All three literal sequences match the native sequence. Nondetection does not prove exact loss of67 residues. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| CloN6 peptide coverage with unresolved proteoform boundaries | has_boundary_limit | Identification, cleavage and assembly limits | The source allows up to19 additional residues from the adjacent tryptic interval. No exact cleavage site, N/C-terminal boundary or unique intact mass is inferred. The five-matching-fragment identification threshold is relayed personal communication, not an independently obtained validation standard. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pCA7 expression attempt in E. coli XL1Blue MRF' | was_scaled_up_in | 1-L TB pCA7 expression and Ni-NTA purification | 1L TB,20C,0.5mM IPTG; methods specify6h after induction. The parallel pCA11 trial used BL21(DE3)/pLysS; the weak positive purification observation belongs to pCA7, not both constructs. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| 1-L TB pCA7 expression and Ni-NTA purification | yielded_unconfirmed_band | Weak65.7-kDa band in impure pCA7 eluates | No overt full-protein induction; a weak65.7-kDa band was seen after Ni-NTA purification with contaminating proteins. It is distinct from the approximately60-kDa band that underwent peptide MS. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Weak65.7-kDa band in impure pCA7 eluates | has_identity_limit | Identification, cleavage and assembly limits | The thesis explicitly requires MS identity confirmation and expression/purification optimization. No pure active CloN6, quantitative yield, catalytic turnover or cofactor stoichiometry is established. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pACYCisc coexpression trial | showed_source_assigned_expression_of | Source-assigned E. coli IscS support protein | FigureIII.3b assigns an approximately45-kDa band to IscS; no independent exact-sequence or binding-partner identity. This is a support protein, not a native clorobiocin-cluster subunit. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pACYCisc coexpression trial | showed_source_assigned_expression_of | Source-assigned E. coli IscU support protein | FigureIII.3b assigns an approximately14-kDa band to IscU; no independent exact-sequence or binding-partner identity. This is a support protein, not a native clorobiocin-cluster subunit. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| pACYCisc coexpression trial | showed_source_assigned_expression_of | Source-assigned E. coli HscB support protein | FigureIII.3b assigns an approximately27-kDa band to HscB; no independent exact-sequence or binding-partner identity. This is a support protein, not a native clorobiocin-cluster subunit. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 68-75: HPDLDVER | TableVII.1 reports detected mass 980.57 and calculated Mr 979.56; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues68-75. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 175-192: VETTGFSHKPATDYNNVR | TableVII.1 reports detected mass 2036.25 and calculated Mr 2035.24; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues175-192. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 281-294: MHTYLDAVHDLGYR | TableVII.1 reports detected mass 1691.00 and calculated Mr 1690.00; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues281-294. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 338-351: GTYTMAEMEEWIPR | TableVII.1 reports detected mass 1713.97 and calculated Mr 1712.97; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues338-351. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 359-373: GVMIWFFIGMPYQDR | TableVII.1 reports detected mass 1860.14 and calculated Mr 1859.13; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues359-373. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 378-388: DTVTYSEHLIR | TableVII.1 reports detected mass 1333.79 and calculated Mr 1332.79; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues378-388. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 433-442: QAMVEPLWYR | TableVII.1 reports detected mass 1292.76 and calculated Mr 1291.75; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues433-442. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 455-466: GIQDVSYEAIER | TableVII.1 reports detected mass 1379.8 and calculated Mr 1378.8; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues455-466. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 495-508: LLAEMDHALSLDGR | TableVII.1 reports detected mass 1540.94 and calculated Mr 1539.93; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues495-508. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| Gel-excised band trypsin LC-MS analysis | detected_peptide | Detected CloN6 peptide 543-555: WFDDVTVPEEMIR | TableVII.1 reports detected mass 1636.95 and calculated Mr 1635.94; units and ion-charge convention are not specified in that table. literal sequence maps exactly to native residues543-555. These table entries are not an independently recalibrated mass spectrum or evidence of full-length protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.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, 23 edges
Native deletion and cassette-retaining tag insertion, with six host-specific titers. Review #569. Native identity, experimental preparations, observations and inferred mechanisms remain distinct. This partial curation does not change compound identity, source-owned fields, earlier graphs or SEEDED status.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native cloN6 protein reference | is_native_reference_for_targeted_locus_in | Native-producer delta-cloN6-67 mutant | Native DS12.976 gene inactivation is distinct from the heterologous tag-insertion experiment. Westrich2003 primary abstract supports PCR targeting and product structures; the later thesis names the delta-cloN6-67 culture. Exact deletion endpoints are not invented. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| clo-BG1 intact-cluster cosmid | was_modified_to_make | clo-CA7 cassette-retaining N-His6 insertion cosmid | Double-joint PCR and lambda-RED recombination insert an N-terminal His6 tag with aac(3)IV from pUG019. This cassette lacks FRT sites and oriT and was retained for selection, not excised. The source reports sequencing confirmation, but sequence files were not obtained. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native cloN6 protein reference | is_native_reference_for_targeted_locus_in | clo-CA7 cassette-retaining N-His6 insertion cosmid | The modified construct is not native Q8GHB6. | https://www.uniprot.org/uniprotkb/Q8GHB6 |
| clo-CA7 cassette-retaining N-His6 insertion cosmid | has_literal_primer_conflict | Tag-insertion primer-table/method disagreement | Table forward CAACAAATAGTTTGGATGTCCCAGTGAAATTGAACGCGGAGGTCTAGAGCAGTACCAGCGTACGGCCC; methods forward CAACAAATAGTTTGGATGTCCCAGTGAAATTGAAGCGGAGGTCTAGAGCAGTACCAGCGTACGGCCC; middle TCGATTGGCTGAGCTCATAAACTAGTATGCGGGGTTCTCATCATCATCATCATCATTTC; reverse GTCCCGGAAGTCGTACACGCTGGGCGCATGTAGCAGGAAATGATGATGATGATGATGAGA. The table36-nt arm matches25251..25286; methods lack one C and call the arm35nt. The reverse39-nt arm matches25295..25333 on the opposite strand. No complete cassette/fusion sequence is reconstructed without an inspected sequenced vector. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1 |
| clo-CA7 cassette-retaining N-His6 insertion cosmid | was_integrated_in | S. coelicolor M512 with integrated clo-CA7 | PEG-mediated protoplast transformation; apramycin and kanamycin selection. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 with integrated clo-CA7 | has_genotyping_readout | clo-CA7 integration Southern readout | FigureIII.6-2C XhoI pattern contrasts the parent6.6-kb fragment with5.2and2.4kb for the insertion. One integration-mutant lane is displayed, not four displayed replicate genotypes. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native-producer delta-cloN6-67 mutant | was_cultured_for | Production-culture extraction and HPLC analysis | Native mutant experiment. YMG preculture2days30C180rpm, cornstarch2-3days33C210rpm, then80mL distillers-solubles medium7-10days33C210rpm are the cited production method. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 with integrated clo-CA7 | was_cultured_for | Production-culture extraction and HPLC analysis | Four integration mutants were cultivated; the text does not specify four quantitative replicates per reported titer or how they were summarized. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Clorobiocin chemical standard in FigureIII.7A | was_analytical_reference_in | Production-culture extraction and HPLC analysis | FigureIII.7A is clorobiocin standard, not a wild-type culture. PanelB is M512/clo-CA7 and panelC is native delta-cloN6-67. Extraction:1mL culture acidified to pH4, twice1mL ethyl acetate,100-300uL methanol and50-100uL injection; RP18 HPLC at340nm. Individual-congener response factors, uncertainty and quantitative detection limits are not supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native-producer delta-cloN6-67 mutant | accumulated_structurally_assigned_product | Source-assigned novclobiocin109 | Westrich2003 reports loss of the pyrrole5-methyl group; NMR/MS confirm metabolite structures. No exact chemical identifier is assigned from this abstract. | DOI:10.1002/cbic.200300609
|
| Native-producer delta-cloN6-67 mutant | accumulated_structurally_assigned_product | Source-assigned novclobiocin110 | Reported isomer has pyrrole-2-carboxyl at sugar2-OH instead of3-OH; not an independently grounded complete stereostructure here. | DOI:10.1002/cbic.200300609
|
| Native-producer delta-cloN6-67 mutant | accumulated_structurally_assigned_product | Source-assigned novclobiocin104 | Reported derivative lacks the whole pyrrole unit. Antibacterial activity and target mechanisms are not copied into this biosynthesis graph. | DOI:10.1002/cbic.200300609
|
| S. coelicolor M512 with integrated clo-CA7 | did_not_show_detectable_production_of | Source-assigned clorobiocin | Under the reported culture/extraction/HPLC conditions, not a universal absence or a quantitative zero. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native-producer delta-cloN6-67 mutant | supports_genetic_involvement_in | Genetically supported pyrrole-methylation involvement | Loss of methylated product and altered congener profile support involvement in methylation but not purified CloN6 catalysis, cofactor dependence or reaction timing. | DOI:10.1002/cbic.200300609 |
| S. coelicolor M512 with integrated clo-CA7 | has_interpretation_limit | Cassette, host and causal-interpretation limits | Retained aac(3)IV, altered expression and host differences prevent attributing the profile solely to direct His-tag inactivation. The source interpretation of impaired CloN7 acyl transfer is not an isolated-enzyme rate or exclusion of polar effects. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Genetically supported pyrrole-methylation involvement | has_unresolved_order_and_assembly | Proposed coordinated CloN1/CloN6/CloN7 process | The early late-methylation interpretation and later proposed pyrrolyl-2-carboxy-S-CloN1 substrate/order are not settled by this deletion. The thesis proposes coordinated CloN1/N6/N7 action, not a directly observed physical complex or measured stoichiometry. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Proposed coordinated CloN1/CloN6/CloN7 process | has_evidence_limit | Cassette, host and causal-interpretation limits | No activity-tested pure CloN6 preparation or demonstrated stable assembly is supplied by these experiments. CouN7 studies are not silently relabeled as native CloN7 biochemistry. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native-producer delta-cloN6-67 mutant | had_reported_hplc_titer_for | Source-assigned novclobiocin104 | 61.2 ug/mL under the cited culture/extraction/HPLC method. No quantitative replicate count, uncertainty, detection limit or statistical ratio equivalence supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native-producer delta-cloN6-67 mutant | had_reported_hplc_titer_for | Source-assigned novclobiocin109 | 11.1 ug/mL under the cited culture/extraction/HPLC method. No quantitative replicate count, uncertainty, detection limit or statistical ratio equivalence supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| Native-producer delta-cloN6-67 mutant | had_reported_hplc_titer_for | Source-assigned novclobiocin110 | 3.45 ug/mL under the cited culture/extraction/HPLC method. No quantitative replicate count, uncertainty, detection limit or statistical ratio equivalence supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 with integrated clo-CA7 | had_reported_hplc_titer_for | Source-assigned novclobiocin104 | 24.2 ug/mL under the cited culture/extraction/HPLC method. No quantitative replicate count, uncertainty, detection limit or statistical ratio equivalence supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 with integrated clo-CA7 | had_reported_hplc_titer_for | Source-assigned novclobiocin109 | 3.9 ug/mL under the cited culture/extraction/HPLC method. No quantitative replicate count, uncertainty, detection limit or statistical ratio equivalence supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
| S. coelicolor M512 with integrated clo-CA7 | had_reported_hplc_titer_for | Source-assigned novclobiocin110 | 0.86 ug/mL under the cited culture/extraction/HPLC method. No quantitative replicate count, uncertainty, detection limit or statistical ratio equivalence supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49061/pdf/Diss_C.Anderle.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 17 nodes, 22 edges
CloY deletion and host/heterologous MbtH-like rescue in S.coelicolor, with exact native references separated from experimental constructs. Partial phenotypes and source disagreements are retained. Evidence review #670; SEEDED status unchanged.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| S.coelicolor M512 with intact cloBG01 | was_assayed_in | Culture-extract HPLC comparison with clorobiocin standard | Heterologous M512 is described as an M145 derivative, not the natural clorobiocin producer. Culture extracts were analyzed by RP-HPLC at340nm with an authentic clorobiocin standard. No quantitative uncertainty or detection limit is invented. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| CloY native sequence reference | is_native_reference_for_gene_targeted_in | M512 with cloY-deleted cloMW16 | cloBG01 carries the clorobiocin cluster; cloMW16 is the reported in-frame scar derivative after removing the initial resistance cassette in cloMW15. This is neither a native DS12.976 knockout nor a verified scarless allele. The full historical construct sequence was not independently inspected. | https://www.uniprot.org/uniprotkb/Q8GHC8 |
| M512 with cloY-deleted cloMW16 | retained_reduced_production_of | Source-assigned clorobiocin | HPLC gives average12 mg/L versus54 mg/L for the intact-cluster control, about22% retained under these conditions. Text later uses equivalent ug/mL. Four individual integration mutants are described; no statistical equivalence, complete abolition or universal dispensability follows. Congener peaks remain distinct. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| M512 with cloY-deleted cloMW16 | was_compared_with | MW03 with cloY-deleted cloMW16 and host cdaX/cchK replacements | Host CDA-ORFX/SCO3218 and cchK/SCO0489 are separately cassette-replaced in MW03 before introduction of cloMW16. Their host roles are not native clorobiocin-cluster membership. PCR supports targeted replacements, not whole-genome identity or absence of every possible compensating protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| CdaX / CDA-ORFX / SCO3218 native sequence reference | is_native_reference_for_host_gene_targeted_in | MW03 with cloY-deleted cloMW16 and host cdaX/cchK replacements | The study names CDA-ORFX/SCO3218 in M512. The exact sequence reference is A3(2)/M145, not an independently sequenced MW03 derivative. | https://www.uniprot.org/uniprotkb/Q9Z388 |
| CchK / SCO0489 native sequence reference | is_native_reference_for_host_gene_targeted_in | MW03 with cloY-deleted cloMW16 and host cdaX/cchK replacements | The study names cchK/SCO0489. Exact native reference grounding does not make the reference protein the cassette-disrupted allele or verify an entire M512 genome. | https://www.uniprot.org/uniprotkb/Q9RK17 |
| MW03 with cloY-deleted cloMW16 and host cdaX/cchK replacements | retained_low_production_of | Source-assigned clorobiocin | Reported HPLC production averages0.3 ug/mL versus roughly50 ug/mL for the intact-cluster control in this comparison, described as about99% reduction. Keep this comparison separate from54/12 and the empty-vector control; do not replace residual production with zero or invent a common replicate count. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| Triple mutant with empty pUWL201 | had_low_production_of | Source-assigned clorobiocin | The empty-vector control for complementation produced less than0.5 ug/mL, not an exact0.5 or a zero. HPLC readout, not purified-protein catalysis. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| MW03 with cloY-deleted cloMW16 and host cdaX/cchK replacements | has_source_annotation_limits | Unresolved source construct-size and mutant-count labels | The source states218 bp for cloY while the deposited CDS is216 nt including stop. It describes an18-bp scar but gives421/258-bp intact/deletion PCR labels; these do not fully specify a verified deletion allele. CDA cassette PCR labels differ between Figure7 (2189bp) and Figure8 (2160bp). Figure5 shows four triple-mutant lanes, whereas the later text/Figure8 describes three. These are retained source discrepancies, not silently corrected construct data. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1 |
| CloY native sequence reference | is_native_reference_for_gene_expressed_in | Triple mutant complemented with CloY via pMW01 | The source reports the corresponding gene in pMW01 under ermE* control on pUWL201 and insert sequencing. Native reference identity is not independent sequencing of the complete experimental plasmid or its expressed product. CouY is from the coumermycin cluster; CdaX/CchK are host-cluster homologs, not additional native clorobiocin BGC proteins. | https://www.uniprot.org/uniprotkb/Q8GHC8 |
| Triple mutant complemented with CloY via pMW01 | partially_restored_production_of | Source-assigned clorobiocin | Reported culture-extract HPLC clorobiocin titer 8 ug/mL. Three independent transformants per construct are described. No error bars, statistical significance or complete baseline recovery are inferred; Figure10 separates clorobiocin from accompanying congeners. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| Triple mutant complemented with CloY via pMW01 | supports_context_bounded | Context-dependent MbtH-like functional compensation | Restoration above the empty-vector condition supports genetic functional compensation under the tested expression context. It is not measured protein binding, a four-subunit complex or an exact catalytic reaction. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| CchK / SCO0489 native sequence reference | is_native_reference_for_gene_expressed_in | Triple mutant complemented with CchK / SCO0489 via pMW02 | The source reports the corresponding gene in pMW02 under ermE* control on pUWL201 and insert sequencing. Native reference identity is not independent sequencing of the complete experimental plasmid or its expressed product. CouY is from the coumermycin cluster; CdaX/CchK are host-cluster homologs, not additional native clorobiocin BGC proteins. | https://www.uniprot.org/uniprotkb/Q9RK17 |
| Triple mutant complemented with CchK / SCO0489 via pMW02 | partially_restored_production_of | Source-assigned clorobiocin | Reported culture-extract HPLC clorobiocin titer 4.1 ug/mL. Three independent transformants per construct are described. No error bars, statistical significance or complete baseline recovery are inferred; Figure10 separates clorobiocin from accompanying congeners. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| Triple mutant complemented with CchK / SCO0489 via pMW02 | supports_context_bounded | Context-dependent MbtH-like functional compensation | Restoration above the empty-vector condition supports genetic functional compensation under the tested expression context. It is not measured protein binding, a four-subunit complex or an exact catalytic reaction. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| CdaX / CDA-ORFX / SCO3218 native sequence reference | is_native_reference_for_gene_expressed_in | Triple mutant complemented with CdaX / CDA-ORFX / SCO3218 via pMW03 | The source reports the corresponding gene in pMW03 under ermE* control on pUWL201 and insert sequencing. Native reference identity is not independent sequencing of the complete experimental plasmid or its expressed product. CouY is from the coumermycin cluster; CdaX/CchK are host-cluster homologs, not additional native clorobiocin BGC proteins. | https://www.uniprot.org/uniprotkb/Q9Z388 |
| Triple mutant complemented with CdaX / CDA-ORFX / SCO3218 via pMW03 | partially_restored_production_of | Source-assigned clorobiocin | Reported culture-extract HPLC clorobiocin titer 5.1 ug/mL. Three independent transformants per construct are described. No error bars, statistical significance or complete baseline recovery are inferred; Figure10 separates clorobiocin from accompanying congeners. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| Triple mutant complemented with CdaX / CDA-ORFX / SCO3218 via pMW03 | supports_context_bounded | Context-dependent MbtH-like functional compensation | Restoration above the empty-vector condition supports genetic functional compensation under the tested expression context. It is not measured protein binding, a four-subunit complex or an exact catalytic reaction. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| CouY native sequence reference | is_native_reference_for_gene_expressed_in | Triple mutant complemented with CouY via pMW04 | The source reports the corresponding gene in pMW04 under ermE* control on pUWL201 and insert sequencing. Native reference identity is not independent sequencing of the complete experimental plasmid or its expressed product. CouY is from the coumermycin cluster; CdaX/CchK are host-cluster homologs, not additional native clorobiocin BGC proteins. | https://www.uniprot.org/uniprotkb/Q9F8V3 |
| Triple mutant complemented with CouY via pMW04 | partially_restored_production_of | Source-assigned clorobiocin | Reported culture-extract HPLC clorobiocin titer 5.3 ug/mL. Three independent transformants per construct are described. No error bars, statistical significance or complete baseline recovery are inferred; Figure10 separates clorobiocin from accompanying congeners. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| Triple mutant complemented with CouY via pMW04 | supports_context_bounded | Context-dependent MbtH-like functional compensation | Restoration above the empty-vector condition supports genetic functional compensation under the tested expression context. It is not measured protein binding, a four-subunit complex or an exact catalytic reaction. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| Context-dependent MbtH-like functional compensation | has_rescue_limit | Partial rescue rather than restoration of the intact-cluster baseline | The thesis explicitly states that complementation did not fully restore the original production level. Promoter/copy-number explanations are hypotheses, not measured protein abundance or mechanistic proof. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 7 nodes, 6 edges
Congener feeding localizes a genetic defect without identifying a CloY catalytic reaction or restoring native clorobiocin. Novclobiocin102 remains distinct. Review #670.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| MW03(cloMW16) triple-mutant feeding experiment | was_supplemented_with | 3-amino-4,7-dihydroxy-8-methylcoumarin feed | The supplied aminocoumarin has C8 methyl, whereas clorobiocin has chlorine at that position. This is a heterologous culture feeding experiment, not a purified CloY substrate assay or unchanged native precursor. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| MW03(cloMW16) triple-mutant feeding experiment | produced_after_feeding | Source-assigned novclobiocin102, a C8-methyl congener | HPLC detected source-assigned novclobiocin102 at5.2 ug/mL; the source reports LC-MS confirmation and AppendixVII shows the corresponding LC-MS/MS panel. The congener is not equated with this owner or assigned an independently verified exact chemical identifier. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| MW03(cloMW16) triple-mutant feeding experiment | retained_low_production_of | Residual source-assigned clorobiocin | The source reports average0.3 ug/mL clorobiocin with a separate small Figure11 peak. Feeding rescue of congener production is not restoration of native clorobiocin. One prose occurrence says cloMW6; methods, heading and figure identify cloMW16. This label discrepancy is not a distinct verified strain. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| Source-assigned novclobiocin102, a C8-methyl congener | supports_bypass_of_defect_in | Efficient aminocoumarin-moiety formation | Chemical complementation supports impaired aminocoumarin supply while other assembly activities remain possible in this mutant context. It does not isolate the molecular action of CloY or prove every downstream rate is unchanged. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
| CloY native sequence reference | is_genetically_implicated_in | Efficient aminocoumarin-moiety formation | The source combines deletion/rescue and feeding evidence to implicate MbtH-like function in efficient aminocoumarin formation. CloY is a PROTEIN reference here, not an assigned catalytic enzyme step. Host compensation prevents an inference that CloY alone is always essential. | https://www.uniprot.org/uniprotkb/Q8GHC8 |
| Efficient aminocoumarin-moiety formation | has_evidence_limit | No isolated-enzyme reaction, physical assembly or universal requirement | The2007 source leaves catalytic, regulatory and protein-interaction mechanisms unresolved. Failure to observe pyrrole-deficient novclobiocin104 is condition-bounded. No direct CloH/CloY binding, stoichiometry, reaction order, purified kinetics or later-paper mechanism is inferred from these genetics. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49097/pdf/Dissertation_Wolpert.pdf?sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 13 nodes, 14 edges
Native CloH grounding and host-conditioned CloH/CloY adenylation evidence, with literal initiation recoding, controls and a bounded kinetic fit. Review #672.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native CloH sequence reference | is_native_reference_for | pBB26 His6-CloH from the ybdZ-deletion host | The source names AAN65224 and amplifies cloH from a cluster cosmid into pET28a as pBB26, an N-terminal His6 fusion. BL21(DE3) carries the ybdZ cassette replacement and pSU20_sfp. The source reports full-gene sequencing, not an independently inspected complete plasmid, tag junction or native DS12.976 preparation. | https://www.uniprot.org/uniprotkb/Q8GHC7 |
| Native CloH sequence reference | is_native_reference_for | pBB26 His6-CloH from the intact-ybdZ host | The comparison uses the corresponding pBB26 preparation from BL21(DE3) with intact ybdZ and pSU20_sfp. Native sequence identity does not establish equivalent active fractions or prove the absence/presence of every copurifying partner. | https://www.uniprot.org/uniprotkb/Q8GHC7 |
| Native CloH sequence reference | was_compared_with | Printed CloH primer model, not a complete plasmid | The visually checked printed NdeI/XhoI primers predict an1823-nt amplicon including restriction tails. Native CDS nucleotide1 changes T to A, TTG to ATG initiation, but the CDS-aware 600-aa protein core is unchanged. This is an insert model, not complete historical construct sequencing. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| Native CloY sequence reference | is_native_reference_for | Separately prepared pBB37 CloY with reported tag processing | pBB37 in Rosetta2(DE3) supplies the separately purified partner. Methods report removal of its N-His tag before gel filtration; the exact post-cleavage scar is not independently determined. This preparation is distinct from the untagged CloY expressed with CloH in pBB35. | https://www.uniprot.org/uniprotkb/Q8GHC8 |
| pBB26 His6-CloH from the ybdZ-deletion host | was_compared_with | pBB26 His6-CloH from the intact-ybdZ host | Reported purification yields are2.83 versus11.3 mg/L culture, respectively; most deletion-host CloH was insoluble. These are preparation yields, not native pathway titers, catalytic rates or quantified active fractions. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB26 His6-CloH from the ybdZ-deletion host | was_assayed_in | L-tyrosine-dependent ATP-PPi exchange assay | The source measures L-tyrosine-dependent ATP-[32P]PPi exchange for5min at30C. Standard enzyme:partner is1:1.2 when partner is present; kinetic and titration experiments vary substrate or partner separately. General methods state two independent reactions; Figure3 explicitly gives six for CloH only. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB26 His6-CloH from the ybdZ-deletion host | retained_without_added_cloy | Low unassisted CloH activity in the deletion-host preparation | The source reports very low activity without added partner; Figure3 labels CloH at0.014 nkat/mg protein. This is not a zero or a universal MbtH requirement. The99.0-99.8% range in discussion spans several enzymes, not a CloH-specific value. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB26 His6-CloH from the ybdZ-deletion host | was_combined_with_partner_in | Reconstituted CloH plus CloY exchange assay | The mixture contains separately purified deletion-host CloH and CloY. The native accession is not assigned to a whole engineered preparation. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Separately prepared pBB37 CloY with reported tag processing | stimulated_cloh_in | Reconstituted CloH plus CloY exchange assay | Adding CloY made the tyrosine-dependent exchange activity readily detectable. The graph does not infer an independently measured binding constant from activation. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| MbtH-only exchange control | constrains_interpretation_of | Reconstituted CloH plus CloY exchange assay | MbtH-like proteins alone showed no exchange activity in the reported control. No numerical detection limit or independently fitted zero is supplied. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Reconstituted CloH plus CloY exchange assay | yielded | CloH/CloY L-tyrosine kinetic fit | At enzyme:partner1:1.2, nonlinear regression with varied L-tyrosine gives Km186.3 +/-16.9 uM in FigureS3 (186uM rounded in Table1), kcat0.497 s^-1 and efficiency2670 s^-1 M^-1. The inspected methods/caption do not define the plus/minus statistic. Values are reported, not refitted or reassigned from NovH. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Low unassisted CloH activity in the deletion-host preparation | did_not_support_a_separate_fit_for | CloH/CloY L-tyrosine kinetic fit | The authors state that CloH-alone activity was too low for kinetic investigation. Do not infer Km, kcat, an infinite fold change or a zero-rate fit without CloY. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB26 His6-CloH from the intact-ybdZ host | supports_condition_bounded | Suggested YbdZ copurification, not directly quantified | For CloH and Pcza361.18 the text gives unassisted activity at20-30% of the corresponding added-partner condition. It suggests E.coli YbdZ copurification. The range is not separately resolved per enzyme and no exact CloH-YbdZ accession, binding measurement or stoichiometric contamination is established. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| L-tyrosine-dependent ATP-PPi exchange assay | has_evidence_limit | Adenylation readout without measured PCP loading or mature-product flux | ATP-PPi exchange is evidence for the adenylation half-reaction. This study does not directly measure PCP loading, hydroxylation, aminocoumarin release, mature clorobiocin synthesis or pathway flux in the native producer. Sfp coexpression does not substitute for an occupancy measurement. | 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 15 nodes, 18 edges
CloH/CloY association and proposed stoichiometry, distinct coexpression and L383M activity tradeoffs, and computational limits. Review #672.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Separately prepared CloH/CloY mixture | showed | Activity saturation near an equimolar CloY:CloH ratio | Figure4 varies added CloY and shows increasing exchange activity up to about1:1, followed by a plateau. No binding constant, confidence interval or exact physical subunit count is determined from this activity titration. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Separately prepared CloH/CloY mixture | yielded | CloH and CloY bands after analytical gel filtration | FigureS4 is an SDS-PAGE image with source-labelled CloH and CloY bands after analytical gel filtration. Band presence supports copurification; the image is not calibrated densitometry or an absolute stoichiometry assay. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| CloH and CloY bands after analytical gel filtration | supports | Source-supported recombinant CloH/CloY association | The source reports copurification and shows both proteins in the isolated fraction. This is association evidence in the recombinant context, not an atomic structure, an independently measured dissociation constant or native in-cell occupancy. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Activity saturation near an equimolar CloY:CloH ratio | supports_source_proposal_of | Proposed CloH/CloY heterotetramer | Equimolar functional saturation contributes to the proposed1:1 subunit ratio. It does not by itself distinguish a heterodimer from a heterotetramer. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Source-supported recombinant CloH/CloY association | has_proposed_assembly | Proposed CloH/CloY heterotetramer | The source says CloH alone formed dimers and that its gel-filtration difference from the CloH/CloY complex was small. Combined with the activity ratio, this motivates a heterotetramer proposal. The650/208/229-kDa values and Figure5 belong to SimH/SimY and are not CloH measurements. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Native CloH sequence reference | is_native_reference_for_coexpression_in | pBB35 coexpressed N-His6-CloH and untagged CloY preparation | pBB35 uses pETDUET1 to coexpress N-His6-CloH and untagged CloY in the ybdZ-deletion BL21(DE3) host with pSU20_sfp. Native CloH is a sequence reference, not the complete fusion or a guarantee of its active fraction. | https://www.uniprot.org/uniprotkb/Q8GHC7 |
| Native CloY sequence reference | is_native_reference_for_coexpression_in | pBB35 coexpressed N-His6-CloH and untagged CloY preparation | AF329398.1 annotates cloY at 12920..13135(+) as 71-aa MbtH-like protein AAN65223.1 / UniProtKB:Q8GHC8. In pETDuet-1-cloH-cloY, cloY is coexpressed as an untagged partner alongside His6-CloH (whereas solitary pET37b-CloY encodes a C-terminal His8 tag). The native CloY reference node is linked to the coexpression construct and recombinant complex without equating the recombinant preparation with the native protein. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| Native CloH sequence reference | is_native_subunit_reference_for | Source-supported recombinant CloH/CloY association | UniProtKB:Q8GHC7 (AAN65224.1, AF329398.1:13390..15192(+), 600 aa) is the deposited native sequence reference for the CloH subunit of the recombinant His6-CloH/CloY complex copurified from pETDUET1-cloH-cloY (pBB35). | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| Native CloY sequence reference | is_native_subunit_reference_for | Source-supported recombinant CloH/CloY association | UniProtKB:Q8GHC8 (AAN65223.1, AF329398.1:12920..13135(+), 71 aa) is the deposited native sequence reference for the CloY MbtH-like subunit of the recombinant His6-CloH/CloY complex copurified from pETDUET1-cloH-cloY (pBB35). | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| pBB35 coexpressed N-His6-CloH and untagged CloY preparation | has_bounded_construct_model | Printed pBB35 insert-facing sequence model | The visually inspected BamHI/NotI primers model a thrombin-recognition segment LVPRGS followed by native CloH residues2..600, omitting the native initiator from that insert-facing segment. This does not reconstruct the upstream vector tag, complete pBB35, cleavage state or experimentally sequenced full fusion. | https://www.uniprot.org/uniprotkb/Q8GHC7 |
| pBB35 coexpressed N-His6-CloH and untagged CloY preparation | provided_copurification_evidence_for | Source-supported recombinant CloH/CloY association | Ni-affinity and gel chromatography yielded an active preparation containing the coexpressed tagged CloH and untagged CloY, interpreted by the authors as complex copurification. Reported preparation yield4.8 mg/L is not a subunit ratio or proof that every recovered CloH molecule retained CloY. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB35 coexpressed N-His6-CloH and untagged CloY preparation | showed | Lower activity of the copurified coexpression preparation | The source reports50% less activity than a mixture of separately purified CloH and CloY. Additional CloY raised activity but not above the separate mixture. The10-fold/75-fold coexpression improvements discussed nearby concern CmnO/VioO from another study and are not transferred. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Lower activity of the copurified coexpression preparation | supports_source_hypothesis_of | Suggested loss of CloY during purification | The authors infer that some CloY may have been lost during purification. Neither the lost fraction nor a physical dissociation rate was quantified. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Native CloH sequence reference | is_native_sequence_reference_for_variant | Engineered CloH L383M preparation, not the native reference | Native residue383 is leucine. Modeling the visually checked mutagenic primer against AF329398.1 changes internal CDS nucleotide1147 T to A (TTG to ATG), producing L383M only in the core. This is distinct from the initiation recoding used for expression. The source reports full-gene sequencing and transfer to pET28a; no complete historical mutant plasmid was independently obtained. The mutant has no exact-native accession assignment. | https://www.uniprot.org/uniprotkb/Q8GHC7 |
| Engineered CloH L383M preparation, not the native reference | showed_without_added_cloy | L383M exchange activity without added CloY | Reported ATP-PPi exchange activity0.22 nkat/mg protein,33% of the source NovH comparison. This is not33% of CloH+CloY and is not mature-product yield. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Engineered CloH L383M preparation, not the native reference | showed_with_added_cloy | L383M exchange activity with added CloY | Reported exchange activity2.45 nkat/mg protein,36% of native-sequence CloH with CloY. Increased unassisted activity therefore comes with reduced partner-assisted activity, not an unconditional improvement or NovH conversion. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| L383M exchange activity without added CloY | is_consistent_with_but_does_not_prove | PheA-based conformational hypothesis | The2011 authors modeled CloH/NovH on PheA (PDB1AMU), proposing effects of residue383 on Y397/E316 and Mg-related geometry. The mutation supports a functional role but does not establish the proposed atomic contacts or exclude alternative mechanisms. Their structure-availability statement is historical. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Source-supported recombinant CloH/CloY association | has_unresolved_structural_explanation | PheA-based conformational hypothesis | Docking gave several possible arrangements and the authors explicitly left complex structure speculative. Their conformational hypothesis does not establish direct CloY-substrate contacts or PCP-domain rotation. | 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, 14 edges
Cognate and noncognate MbtH activation of CloH in vitro, with native references separated from processed/synthetic partners and per-panel normalization. Review #672.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native CloH sequence reference | is_native_reference_for | Deletion-host recombinant CloH preparation | Figure6 uses recombinant CloH from the ybdZ-deletion host. Native Q8GHC7 does not denote the engineered His6 fusion. | https://www.uniprot.org/uniprotkb/Q8GHC7 |
| Deletion-host recombinant CloH preparation | was_assayed_in | Four-partner CloH cross-activation panel | ATP-PPi exchange compares CloY, CdaX, SimY and Orf1van at enzyme:partner 1:1.2. Figure6 normalizes each enzyme panel independently to its CloY condition. No exact percentages are newly digitized or compared across enzymes as if their absolute denominators were shared. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| CloY native sequence reference | is_native_reference_for | pBB37-derived CloY preparation | 71-aa native AAN65223.1/Q8GHC8; the cluster-cosmid-derived partner is cognate at the source-pathway level. The prepared protein remains separate from both the native reference and untagged pBB35 coexpression product. | https://www.uniprot.org/uniprotkb/Q8GHC8 |
| pBB37-derived CloY preparation | stimulated_cloh_in | Four-partner CloH cross-activation panel | The source reports stimulation of CloH by this partner relative to no added MbtH. Partners were expressed in Rosetta2(DE3) with N-His tags; methods describe tag removal before gel filtration. Neither that processing statement nor stimulation establishes an exact cleavage scar, a measured binding constant or native physiological pairing. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| CdaX / SCO3218 native sequence reference | is_native_reference_for | pBB32-derived CdaX / SCO3218 preparation | 71-aa CAB38589.1 matches Q9Z388 and AL939115.1:183050..183265(-) CDS-aware translation. The GTG start is initiator methionine. UniProt names A3(2)/M145, taxon100226, whereas the source amplified cdaX from M512. This reference does not independently sequence the experimental strain/construct. | https://www.uniprot.org/uniprotkb/Q9Z388 |
| pBB32-derived CdaX / SCO3218 preparation | stimulated_cloh_in | Four-partner CloH cross-activation panel | The source reports stimulation of CloH by this partner relative to no added MbtH. Partners were expressed in Rosetta2(DE3) with N-His tags; methods describe tag removal before gel filtration. Neither that processing statement nor stimulation establishes an exact cleavage scar, a measured binding constant or native physiological pairing. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| SimY native sequence reference | is_native_reference_for | pBB28-derived SimY preparation | The sim-cluster-cosmid partner maps to native Q9F5J2. AF322256.1 AAL15602.1 translates to70 aa and matches the prior source-named AAG34186.1 comparison. Native taxon1890 is S.antibioticus. The source reports69 aa, so no exact experimental-length correction is assumed. | https://www.uniprot.org/uniprotkb/Q9F5J2 |
| pBB28-derived SimY preparation | stimulated_cloh_in | Four-partner CloH cross-activation panel | The source reports stimulation of CloH by this partner relative to no added MbtH. Partners were expressed in Rosetta2(DE3) with N-His tags; methods describe tag removal before gel filtration. Neither that processing statement nor stimulation establishes an exact cleavage scar, a measured binding constant or native physiological pairing. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Orf1van native sequence reference | is_native_reference_for | pBB34-derived Orf1van preparation | Native AAL90876.1 is69 aa, matching AF486630.1:720..929(+) in Amycolatopsis orientalis taxon31958. The source reports70 aa and uses a commercially synthesized codon-optimized gene. No independent full synthetic construct or exact UniProt mapping is claimed; retain the source-named NCBI identifier. | https://www.ncbi.nlm.nih.gov/protein/AAL90876.1 |
| pBB34-derived Orf1van preparation | stimulated_cloh_in | Four-partner CloH cross-activation panel | The source reports stimulation of CloH by this partner relative to no added MbtH. Partners were expressed in Rosetta2(DE3) with N-His tags; methods describe tag removal before gel filtration. Neither that processing statement nor stimulation establishes an exact cleavage scar, a measured binding constant or native physiological pairing. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| Four-partner CloH cross-activation panel | has_partner_scope_limit | Noncognate functional activation, not native-cluster membership | CloY gave the highest displayed CloH activity. CdaX, SimY and Orf1van are noncognate in-vitro partners, not newly assigned native clorobiocin BGC components. CchK/CouY rescue in the separate genetic study does not establish that they were tested in this2011 purified-protein panel. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1
|
| pBB28-derived SimY preparation | has_source_discrepancy | Partner length and tag-stage discrepancies | The source lists69-aa SimY while the native reference has70 aa. FigureS2 labels N-His6-SimY and9.4kDa despite general methods describing tag removal. A different preparation stage is possible, but the activation-assay tag state and scar are not independently established. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| pBB34-derived Orf1van preparation | has_source_discrepancy | Partner length and tag-stage discrepancies | The paper lists70-aa Orf1van but the source-named native protein/CDS gives69 aa. No initiator-removal explanation, label swap or synthetic sequence repair is invented. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/handle/10900/49604/pdf/Boll_2011_online.pdf?isAllowed=y&sequence=1 |
| Orf1van native 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.
Bioactivities 2
Entries preserve source qualifiers, targets and evidence. Repeated assay names or values are not independent replications; inspect source detail for differences.
| Assay | Result | Target enzyme | Source detail |
|---|---|---|---|
| Experimentally measured binding affinity data (Kd) for protein-ligand complexes derived from PDB | KD 0.0012 uM; ACTIVE | Not recorded | PMID:12044152
|
| Experimentally measured binding affinity data derived from PDB | ACTIVE | Not recorded | PMID:12044152
|
Elsewhere in the fleet
- AntibioticMech — antibioticmech:aro-e44e701df2 (same structure, same inchikey)
Explore across X-Mech
AntibioticMech (clorobiocin) ↗
MediaIngredientMech (naturalproductmech:mibig-619480541f) ↗
TaxonMech: Streptomyces roseochromogenus subsp. oscitans DS 12.976 (NCBITaxon:1352936) ↗
PathwayMech (mibig:BGC0000832) ↗
Provenance
| Source concept | Source | Version |
|---|---|---|
| BGC0000832 | MIBIG | 4 |
This page is generated from data/natural_products/shikimates_and_phenylpropanoids/clorobiocin.yaml, which is generated from the committed inventories. Neither is edited by hand.