gallate degradation I

MetaCyc:GALLATE-DEGRADATION-II-PWY

Four-step aerobic gallate cleavage to pyruvate and oxaloacetate through keto-4-oxalomesaconate, the E,E enol and stereochemically unspecified carboxyhydroxyoxoadipate. The exact MetaCyc accession has suffix II but displays gallate degradation I. Formal reaction structures control the E,E assignment despite a conflicting Z,Z narrative link. KTGAL repaired GalT, KT2440 reporter constructs, historical native preparations and recombinant assays remain distinct. Source associations do not establish this complete route in every component organism. SYK-6 DesZ and LigAB oxygen-consumption assays show alternative gallate turnover without resolving the immediate product; PCA-specific LigAB regulator edges do not imply gallate-flux regulation.

Pathway type
aromatic compound catabolism
Organism scope
Not recorded

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Pathway network

148 nodes · 130 asserted directed edges

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Pathway network: gallate degradation I 148 nodes · 130 asserted directed edges. Every arrow links to its evidence table row; nodes link to their component details. Isolated declared components are retained. RHEA:28928 — part_of → MetaCyc:GALLATE-DEGRADATION-II-PWY; Formal native membership; accession suffix II is distinct from displayed name I.; Evidence: MetaCyc:GALLATE-DEGRADATION-II-PWYpart of RHEA:28928 — has_input → CHEBI:16918; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:GALLATE-DIOXYGENASE-RXNhas input RHEA:28928 — has_input → CHEBI:15379; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:GALLATE-DIOXYGENASE-RXNhas input RHEA:28928 — has_output → CHEBI:57471; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:GALLATE-DIOXYGENASE-RXNhas output RHEA:28928 — has_output → CHEBI:15378; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:GALLATE-DIOXYGENASE-RXNhas output RHEA:28932 — part_of → MetaCyc:GALLATE-DEGRADATION-II-PWY; Formal native membership; accession suffix II is distinct from displayed name I.; Evidence: MetaCyc:GALLATE-DEGRADATION-II-PWYpart of RHEA:28932 — has_input → CHEBI:57471; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:RXN-9983has input RHEA:28932 — has_output → CHEBI:58142; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:RXN-9983has output RHEA:17403 — part_of → MetaCyc:GALLATE-DEGRADATION-II-PWY; Formal native membership; accession suffix II is distinct from displayed name I.; Evidence: MetaCyc:GALLATE-DEGRADATION-II-PWYpart of RHEA:17403 — has_input → CHEBI:58142; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:RXN-2463has input RHEA:17403 — has_input → CHEBI:15377; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:RXN-2463has input RHEA:17403 — has_output → CHEBI:58075; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:RXN-2463has output RHEA:28936 — part_of → MetaCyc:GALLATE-DEGRADATION-II-PWY; Formal native membership; accession suffix II is distinct from displayed name I.; Evidence: MetaCyc:GALLATE-DEGRADATION-II-PWYpart of RHEA:28936 — has_input → CHEBI:58075; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:RXN-2464has input RHEA:28936 — has_output → CHEBI:15361; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:RXN-2464has output RHEA:28936 — has_output → CHEBI:16452; Selected physiological route orientation; native reversible metadata retained independently.; Evidence: MetaCyc:RXN-2464has output RHEA:28928 — provides_input_for → RHEA:28932; Native order and direct material continuity.; Evidence: MetaCyc:GALLATE-DEGRADATION-II-PWYprovides input for RHEA:28932 — provides_input_for → RHEA:17403; Native order and direct material continuity.; Evidence: MetaCyc:GALLATE-DEGRADATION-II-PWYprovides input for RHEA:17403 — provides_input_for → RHEA:28936; Native order and direct material continuity.; Evidence: MetaCyc:GALLATE-DEGRADATION-II-PWYprovides input for MetaCyc:CPLX-2701 — catalyzes → RHEA:28928; Historical purified Comamonas/Pseudomonas testosteroni protocatechuate 4,5-dioxygenase gallate activity (1972/1982 references). The native complex identity is retained; modern sequence cross-references do not establish the historical assayed sequence.; Evidence: MetaCyc:ENZRXN-11007catalyzes MetaCyc:CPLX-7917 — catalyzes → RHEA:28936; Historical Comamonas CHA aldolase activity in crude or partly purified preparations. The source-native complex and its historical evidence remain distinct from current genome proteins.; Evidence: MetaCyc:ENZRXN-11041catalyzes MetaCyc:CPLX-7918 — catalyzes → RHEA:28928; subject experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) in E. coli BL21(DE3), purified by POROS chromatography; initiating methionine processed. Full terminal construct sequence and any vector-derived residues were not established.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)); Evidence PMID:16030198: appeared to be processed (PMC1196043 Results, Purification of DesB, first paragraph, initiating methionine sentence following the N-terminal sequence); Purified SYK-6 DesB produced in E. coli BL21(DE3) with pETB/pET21a(+). The encoded 418-residue reference has a processed initiating methionine in the assayed preparation. Gallate-dependent oxygen consumption gives Vmax 42.7 ± 2.4 U/mg and Km 66.9 ± 9.3 µM (2005 Table 2). Late acidified GC-MS supports OMA enol but does not alone determine the immediate keto product; native formal chemistry and the independent GalA product work supply that distinction. In the separate 500 µM substrate screen (30 °C, pH 8.5), pyrogallol supported only 0.11 U/mg versus gallate 41.8; its product was not established. Tested negative substrates are conditional assay negatives.; Evidence: MetaCyc:ENZRXN-16936catalyzes MetaCyc:CPLX-7918 — has_cofactor → CHEBI:29033; Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.; Evidence: MetaCyc:ENZRXN-16936has cofactor MetaCyc:G1G01-2697-MONOMER — catalyzes → RHEA:28932; subject experimental construct: 2011 KT2440 GalD expressed from pETGalDc in E. coli BL21(DE3), assayed in partly purified extract. This is separate from the explicitly His6-retaining 2016 GalD preparation.; Evidence PMID:21219457: pETGalDc (2011 publisher Figure 3 caption line 249 and Results line 253; Methods lines 349, 365–366 and 376–377 (construct, BL21(DE3) expression, extract preparation). Retained main-text transcription; original spectra and Table S1 were inaccessible.); KT2440 GalD in partly purified E. coli BL21(DE3)/pETGalDc extract converts keto OMA to the E enol. The 2011 NMR text reports 6.73 ppm and no approximately 7.38 ppm Z-enol signal; original spectra were not visually accessible. This enzymatic acceleration coexists with spontaneous tautomerization; residual growth of the galD mutant is concentration dependent.; Evidence: MetaCyc:ENZRXN-18372, PMID:21219457catalyzes MetaCyc:CPLX-8178 — catalyzes → RHEA:17403; subject experimental construct: 2011 pET29a-derived KT2440 GalB in E. coli BL21(DE3), purified by DEAE and phenyl chromatography. Table 1 reports 244 residues; reference Q88JX8 has 258. Complete insert/termini remain unresolved; the separate 2016 crystal is 240 residues.; Evidence MetaCyc:ENZRXN-18373: The activity has purified heterologous-host evidence for CPLX-8178 linked to PMID21219457. (/ptools-xml/Enzymatic-Reaction/evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]); Evidence PMID:21219457: Table 1 lists GalB as a 244-residue, 27.4-kDa protein in the gal cluster. This does not identify the complete recombinant insert with the independently deposited 258-residue reference. (2011 Table1, galB row: protein size and predicted molecular mass columns); Evidence PMID:21219457: pETGalB (2011 Results GalB publisher line274: E. coli BL21(DE3)(pETGalB); Methods GalB expression and purification); Purified 2011 pET29a-derived GalB from E. coli BL21(DE3) hydrates the E enol, not fresh keto OMA. Primary Km 85 µM and Vmax 76 µmol/min/mg apply to enol hydration, despite the native XML attaching them to CHA. Table 1 reports a 244-residue protein, whereas the deposited reference is 258 residues; the complete insert is unresolved. The 2016 240-residue crystallization construct does not settle this discrepancy. The formal CHA identifier leaves absolute stereochemistry unspecified.; Evidence: MetaCyc:ENZRXN-18373catalyzes MetaCyc:CPLX-8178 — has_cofactor → CHEBI:29105; Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.; Evidence: MetaCyc:ENZRXN-18373has cofactor MetaCyc:CPLX-2781 — catalyzes → RHEA:17403; Historical purified ProH hydratase from phthalate-grown Pseudomonas straminea/ochraceae; source-native identity and reversible activity are retained. No whole-route occurrence is inferred from this single enzyme.; Evidence: MetaCyc:ENZRXN-3364catalyzes MetaCyc:CPLX-2782 — catalyzes → RHEA:28936; Source-native ProA CHA aldolase annotation combines purified native and heterologous-host evidence. These historical preparations are not assumed to equal every current sequence cross-reference.; Evidence: MetaCyc:ENZRXN-3365catalyzes MetaCyc:CPLX-2782 — has_cofactor → CHEBI:18420; Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.; Evidence: MetaCyc:ENZRXN-3365has cofactor MetaCyc:CPLX-2865 — catalyzes → RHEA:28936; Source-native SYK-6 LigK CHA aldolase activity. The historical/current sequence and taxonomy identifiers are not silently equated; its additional oxaloacetate decarboxylation is outside the four formal members.; Evidence: MetaCyc:ENZRXN-3627catalyzes MetaCyc:CPLX-2865 — has_cofactor → CHEBI:18420; Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.; Evidence: MetaCyc:ENZRXN-3627has cofactor MetaCyc:CPLX-2881 — catalyzes → RHEA:28936; Historical purified Pseudomonas putida CHA aldolase, distinct from the sequence-defined KT2440 GalC. The source also discusses HMG specificity; that does not fix CHA absolute stereochemistry.; Evidence: MetaCyc:ENZRXN-3641catalyzes MetaCyc:CPLX-2881 — has_cofactor → CHEBI:29035; Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.; Evidence: MetaCyc:ENZRXN-3641has cofactor MetaCyc:CPLX-2881 — has_cofactor → CHEBI:18420; Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.; Evidence: MetaCyc:ENZRXN-3641has cofactor MetaCyc:CPLX1G01-155 — catalyzes → RHEA:28936; subject experimental construct: 2011 KT2440 GalC expressed in E. coli BL21(DE3) from pETGalC, assayed as crude extract. Historical purified GalC/hexamer annotations do not establish the sequence or assembly of this recombinant preparation.; Evidence PMID:21219457: pETGalC (2011 publisher Results lines 307–309 and Methods lines 349, 365–366 and 380 (construct, BL21(DE3), crude extract, assay and activities). Retained main-text transcription; original Table S1 and Figure S7 were inaccessible.); KT2440 GalC activity is supported by crude E. coli BL21(DE3)/pETGalC extract and a disrupted KTGAL strain. At 200 µM CHA, 1 mM MgCl2, pH 7, 30 °C the source reports 24.5 µmol/min/mg in recombinant extract and 2.1 in KTGAL, with no detected mutant activity. Whole-cluster complementation and polar effects do not prove isolated-gene sufficiency. Historical purified/homohexamer evidence remains a separate source annotation.; Evidence: MetaCyc:ENZRXN1G01-1112catalyzes MetaCyc:CPLX1G01-155 — has_cofactor → CHEBI:29035; Native Mn2+ annotation for the GalC source instance reflects historical purified-enzyme evidence. It does not establish Mn occupancy in the 2011 KT2440 crude recombinant assay, which contained MgCl2.; Evidence: MetaCyc:ENZRXN1G01-1112has cofactor MetaCyc:CPLX-2701 — has_part → MetaCyc:MONOMER-3165; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX-2701has part MetaCyc:CPLX-2701 — has_part → MetaCyc:MONOMER-3166; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX-2701has part MetaCyc:CPLX-2781 — has_part → MetaCyc:MONOMER-3243; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX-2781has part MetaCyc:CPLX-2782 — has_part → MetaCyc:MONOMER-3244; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX-2782has part MetaCyc:CPLX-2865 — has_part → MetaCyc:MONOMER-3469; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX-2865has part MetaCyc:CPLX-2881 — has_part → MetaCyc:MONOMER-3481; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX-2881has part MetaCyc:CPLX-7917 — has_part → MetaCyc:MONOMER-3184; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX-7917has part MetaCyc:CPLX-7918 — has_part → MetaCyc:MONOMER-15119; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX-7918has part MetaCyc:CPLX-8178 — has_part → MetaCyc:G1G01-2699-MONOMER; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX-8178has part MetaCyc:CPLX1G01-155 — has_part → MetaCyc:G1G01-2698-MONOMER; Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.; Evidence: MetaCyc:CPLX1G01-155has part CHEBI:16452 — inhibits → MetaCyc:CPLX-2865; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-10400inhibits MetaCyc:N-ETHYLMALEIMIDE — inhibits → MetaCyc:CPLX-2865; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-10401inhibits CHEBI:16452 — inhibits → MetaCyc:CPLX-2881; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-10724inhibits CHEBI:16793 — inhibits → MetaCyc:CPLX-2865; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-12626inhibits CHEBI:16793 — inhibits → MetaCyc:CPLX-2781; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-12629inhibits CHEBI:16793 — inhibits → MetaCyc:CPLX-2782; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-12638inhibits CHEBI:29105 — inhibits → MetaCyc:CPLX-2781; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-12669inhibits CHEBI:16452 — inhibits → MetaCyc:CPLX-2782; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-8391inhibits MetaCyc:CPD-29 — inhibits → MetaCyc:CPLX-2782; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-8393inhibits MetaCyc:CPD-29 — inhibits → MetaCyc:CPLX-2781; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-9531inhibits MetaCyc:CUSO4 — inhibits → MetaCyc:CPLX-2781; Source assay effect on this catalyst only, not global inhibition of the pathway.; Evidence: MetaCyc:REG-9532inhibits RHEA:28936 — provides_input_for → MetaCyc:TCA; The final aldolase reaction produces oxaloacetate, the metabolite explicitly linked by this native pathway to the prokaryotic TCA pathway. This is a material interface, not an additional formal gallate reaction.; Evidence: MetaCyc:GALLATE-DEGRADATION-II-PWYprovides input for MetaCyc:GALLATE-DEGRADATION-II-PWY — part_of → MetaCyc:PWY-6339; Syringate parent context; no upstream route reactions added to the four formal members.; Evidence: MetaCyc:GALLATE-DEGRADATION-II-PWYpart of MetaCyc:CPLX-8177 — catalyzes → RHEA:28928; subject experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established. The encoded chain is 420 aa; Edman results suggest 419 aa after initiator-Met processing.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); Evidence NCBIProtein:AAN68130.2: The PP_2518 CDS names protein AAN68130.2 and supplies a 420-residue translation beginning MARIIGG. (AE015451.2 CDS complement(2865514..2866776), /locus_tag, /protein_id and /translation.); Evidence PMID:16030014: appeared to be processed. (Results cloning/expression, p.35385; gal2005_methods-successor-web.json L1011-L1019, exact phrase at L1015.); GalA catalyzes gallate ring cleavage in the purified recombinant assay. The 2005 late product analysis alone does not distinguish the immediate keto/enol product; formal equation selection depends on the native/Rhea and 2011 chemistry reconciliation.; Evidence: MetaCyc:ENZRXN-18371, PMID:16030014, PMID:21219457catalyzes MetaCyc:CPLX-8177 — has_cofactor → CHEBI:29033; Native ENZRXN-18371 records Fe2+ as GalA cofactor with the 2005 publication. This native annotation does not fix iron occupancy or imply that other ions fully restore activity. The recombinant pretreatment/rescue observations are separate qualified edges.; Evidence: MetaCyc:ENZRXN-18371has cofactor MetaCyc:CPLX-8177 — has_part → MetaCyc:G1G01-2702-MONOMER; subject experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); The native component relation and the recombinant assembly experiment are joined here with the preparation qualification. The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. Analytical ultracentrifugation found a predominant species representing about 75% of loading protein, with corrected sedimentation coefficient 7.29 ± 0.10 S and equilibrium mass 132 ± 12 kDa. Comparison with calculated monomer mass 47.644 kDa supports a dominant homotrimer. The preparation was not shown to be exclusively trimeric, and the native component slot supplies no stoichiometric coefficient.; Evidence: MetaCyc:CPLX-8177, PMID:16030014has part CHEBI:4735 — inhibits → MetaCyc:CPLX-8177; object experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. 1 mM EDTA left less than 1% of untreated activity. This is an in-vitro exposure result; no cellular flux or specific metal-occupancy mechanism is asserted. The source names EDTA without a salt or protonation specification. CHEBI:4735 is the canonical named parent-acid reference for that reagent; this normalization does not identify the assayed microspecies, supplied salt, charge or stoichiometry.; Evidence: PMID:16030014, CHEBI:4735inhibits CHEBI:30351 — inhibits → MetaCyc:CPLX-8177; object experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. 2 mM 2,2′-dipyridyl abolished detectable activity. This is an in-vitro exposure result; no cellular flux or specific metal-occupancy mechanism is asserted.; Evidence: PMID:16030014inhibits CHEBI:16240 — inhibits → MetaCyc:CPLX-8177; object experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. 1 mM hydrogen peroxide left 15% of untreated activity. This is an in-vitro exposure result; no cellular flux or specific metal-occupancy mechanism is asserted.; Evidence: PMID:16030014inhibits CHEBI:29033 — activates → MetaCyc:CPLX-8177; object experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. After EDTA or dipyridyl pretreatment, 2 mM Fe2+ supplied as FeSO4 restored activity fully following a further 30 min at 4 °C. The two chelator preparations are not reported separately in these outcomes; this conditional rescue is not activation of an untreated enzyme. Metal salt delivery is retained without equating a salt with the ion endpoint. ; Evidence: PMID:16030014activates CHEBI:29035 — activates → MetaCyc:CPLX-8177; object experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. After chelator pretreatment, 2 mM Mn2+ supplied as MnSO4 restored 8% of untreated activity following a further 30 min at 4 °C. The two chelator preparations are not reported separately in these outcomes; this conditional rescue is not activation of an untreated enzyme. Metal salt delivery is retained without equating a salt with the ion endpoint. ; Evidence: PMID:16030014activates CHEBI:29036 — activates → MetaCyc:CPLX-8177; object experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. After chelator pretreatment, 2 mM Cu2+ supplied as CuCl2 restored 9% of untreated activity following a further 30 min at 4 °C. The two chelator preparations are not reported separately in these outcomes; this conditional rescue is not activation of an untreated enzyme. Metal salt delivery is retained without equating a salt with the ion endpoint. ; Evidence: PMID:16030014activates CHEBI:48828 — activates → MetaCyc:CPLX-8177; object experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. After chelator pretreatment, 2 mM Co2+ supplied as CoCl2 restored 5% of untreated activity following a further 30 min at 4 °C. The two chelator preparations are not reported separately in these outcomes; this conditional rescue is not activation of an untreated enzyme. Metal salt delivery is retained without equating a salt with the ion endpoint. ; Evidence: PMID:16030014activates CHEBI:113451 — activates → MetaCyc:CPLX-8177; object experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. Following the 1 mM peroxide pretreatment, 10 mM sodium ascorbate and a further 30 min at 4 °C restored activity to 50% of untreated enzyme. This is partial rescue of the peroxide-exposed preparation. It does not establish constitutive activation or an in-vivo effect.; Evidence: PMID:16030014activates UniProtKB:Q88JX7 — regulates → KEGG:ppu:PP_2515; subject experimental construct: GalR supplied in an additional plasmid copy in P. putida KT2440, with PP_RS13150-RFP on pIK002_T; the promoter-only pIK002A_T control retains endogenous GalR. Readout: 6 h after 1.25 mM gallate in LB/MM. Native target-gene transcription and direct GalR binding were not measured.; Evidence PMID:36724292: extra copy of galR gene (PMC9942251 XML body paragraph ordinal 10 (zero-based 9); experimental comparison in body paragraph ordinal 8 (zero-based 7) and Figure 2B.); Positive transcriptional regulation of the galb promoter/gene context is inferred from the KT2440 RFP reporter comparison. The promoter PP_RS13150 was assayed on pIK002_T versus pIK002A_T: 45/248-fold induction with the extra-copy construct versus 1.7/6.3-fold with the promoter-only vector in LB/MM, measured 6 h after 1.25 mM gallic acid in the source experiment. The promoter-only host retains its endogenous galR; the comparison is an additional plasmid copy, not a galR-null rescue. This does not directly measure native target-gene RNA, exact promoter occupancy, GalR ligand binding, or GalB/GalT protein activation. The reference GalR sequence is independently grounded; the exact 2023 plasmid insert was not sequenced here.; Evidence: PMID:36724292regulates UniProtKB:Q88JX7 — regulates → KEGG:ppu:PP_2519; subject experimental construct: GalR supplied in an additional plasmid copy in P. putida KT2440, with PP_RS13170-RFP on pIK014_T; the promoter-only pIK014A_T control retains endogenous GalR. Readout: 6 h after 1.25 mM gallate in LB/MM. Native target-gene transcription and direct GalR binding were not measured.; Evidence PMID:36724292: extra copy of galR gene (PMC9942251 XML body paragraph ordinal 10 (zero-based 9); experimental comparison in body paragraph ordinal 8 (zero-based 7) and Figure 2B.); Positive transcriptional regulation of the galt promoter/gene context is inferred from the KT2440 RFP reporter comparison. The promoter PP_RS13170 was assayed on pIK014_T versus pIK014A_T: 9.7/72.3-fold induction with the extra-copy construct versus 2.5/45.6-fold with the promoter-only vector in LB/MM, measured 6 h after 1.25 mM gallic acid in the source experiment. The promoter-only host retains its endogenous galR; the comparison is an additional plasmid copy, not a galR-null rescue. This does not directly measure native target-gene RNA, exact promoter occupancy, GalR ligand binding, or GalB/GalT protein activation. The reference GalR sequence is independently grounded; the exact 2023 plasmid insert was not sequenced here.; Evidence: PMID:36724292regulates MetaCyc:CPLX-7916 — has_part → MetaCyc:MONOMER-15116; Native SYK-6 complex component; no coefficient is present in the MetaCyc component slot. This does not assert that unmodified native material was used in the 2013 or 2015 assays.; Evidence: MetaCyc:CPLX-7916has part MetaCyc:CPLX-7916 — has_part → MetaCyc:MONOMER-15117; Native SYK-6 complex component; no coefficient is present in the MetaCyc component slot. This does not assert that unmodified native material was used in the 2013 or 2015 assays.; Evidence: MetaCyc:CPLX-7916has part MetaCyc:MONOMER-15117 — has_cofactor → CHEBI:29033; The issuing UniProt LigB reference annotation identifies Fe2+ as a cofactor. It does not fix occupancy, prove gallate-specific metal rescue, or establish the exact synthetic construct identity.; Evidence: UniProtKB:P22636has cofactor CHEBI:88714 — inhibits → MetaCyc:CPLX-7916; object experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification); Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 2,3,4-trihydroxybenzoic acid reduced PCA-dependent O2 consumption by 95% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.; Evidence: PMID:23977959inhibits CHEBI:18026 — inhibits → MetaCyc:CPLX-7916; object experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification); Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 2,3-dihydroxybenzoic acid reduced PCA-dependent O2 consumption by 36% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.; Evidence: PMID:23977959inhibits CHEBI:16318 — inhibits → MetaCyc:CPLX-7916; object experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification); Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 4-nitrocatechol reduced PCA-dependent O2 consumption by 31% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.; Evidence: PMID:23977959inhibits CHEBI:17254 — inhibits → MetaCyc:CPLX-7916; object experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification); Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 4-methylcatechol reduced PCA-dependent O2 consumption by 28% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.; Evidence: PMID:23977959inhibits CHEBI:141700 — inhibits → MetaCyc:CPLX-7916; object experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification); Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 3-methoxycatechol reduced PCA-dependent O2 consumption by 26% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.; Evidence: PMID:23977959inhibits CHEBI:4735 — inhibits → MetaCyc:CPLX-7916; object experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification); Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). PCA activity retained 65% after 1 h of EDTA at pH 7.5; the dose is not explicit for that time point. After 24 h at 1 or 2 mM EDTA, 35% remained. A separate 1 mM/2 h treatment retained 50%. Readout: 250 µM PCA (P23/P34). Canonical named-parent normalization does not establish the supplied EDTA salt, assayed neutral acid, unique pH microspecies, charge or stoichiometry.; Evidence: PMID:23977959inhibits CHEBI:30351 — inhibits → MetaCyc:CPLX-7916; object experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification); Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). PCA activity retained 50% after 1 h and none after 24 h with 2 mM bipyridine at 4 °C under N2. Readout: 250 µM PCA in 50 mM Tris, pH 7.5 (P23/P34). No gallate assay or reversible physiological effect was demonstrated.; Evidence: PMID:23977959inhibits CHEBI:29036 — inhibits → MetaCyc:CPLX-7916; object experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification); Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). After 1 mM EDTA for 2 h left 50% activity, 2 mM Cu2+ for 1 h lowered residual PCA activity to 8% of untreated. The metal protocol was anaerobic at 4 °C, with a 250 µM PCA readout (P23/P34). Co2+ and Mn2+ did not cause the additional loss. This is not an untreated-enzyme copper effect or a measured gallate-flux effect.; Evidence: PMID:23977959inhibits CHEBI:29033 — activates → MetaCyc:CPLX-7916; object experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification); Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). After 2 mM bipyridine stripping for 24 h, 2 mM FeSO4 for 1 h anaerobically at 4 °C restored only 5% of untreated PCA activity. Longer incubation did not improve rescue; other tested metals did not rescue (P23/P34). This is partial rescue of treated material, not general activation of native LigAB or gallate flux.; Evidence: PMID:23977959activates CHEBI:36241 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)); Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.; Evidence: DOI:10.1016/j.procbio.2021.01.019inhibits CHEBI:16318 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)); Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.; Evidence: DOI:10.1016/j.procbio.2021.01.019inhibits CHEBI:16632 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)); Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.; Evidence: DOI:10.1016/j.procbio.2021.01.019inhibits CHEBI:89886 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)); Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.; Evidence: DOI:10.1016/j.procbio.2021.01.019inhibits CHEBI:132111 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)); Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.; Evidence: DOI:10.1016/j.procbio.2021.01.019inhibits CHEBI:18346 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)); Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.; Evidence: DOI:10.1016/j.procbio.2021.01.019inhibits CHEBI:67380 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)); Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.; Evidence: DOI:10.1016/j.procbio.2021.01.019inhibits CHEBI:16918 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)); In vitro inhibition at high gallate concentrations in the measured substrate range; gallate remains the substrate at lower concentrations. Not a cellular flux claim.; Evidence: DOI:10.1016/j.procbio.2021.01.019inhibits CHEBI:4735 — inhibits → MetaCyc:CPLX-7918; object experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)); 500 µM EDTA for 20 h at 0 °C eliminated detected gallate-dependent oxygen consumption. The oxygen-consumption assay used gallate; this is a treatment-specific in vitro effect, not a physiological metal preference. EDTA is normalized to its named parent without assigning the supplied salt or assay microspecies.; Evidence: PMID:16030198inhibits CHEBI:29033 — activates → MetaCyc:CPLX-7918; object experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)); After EDTA treatment, 1 mM Fe2+ for 5 min on ice restored 119% of untreated activity. The oxygen-consumption assay used gallate; this is a treatment-specific in vitro effect, not a physiological metal preference. EDTA is normalized to its named parent without assigning the supplied salt or assay microspecies.; Evidence: PMID:16030198activates CHEBI:29035 — activates → MetaCyc:CPLX-7918; object experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)); After EDTA treatment, 1 mM Mn2+ for 5 min on ice restored 14% of untreated activity. The oxygen-consumption assay used gallate; this is a treatment-specific in vitro effect, not a physiological metal preference. EDTA is normalized to its named parent without assigning the supplied salt or assay microspecies.; Evidence: PMID:16030198activates CHEBI:29036 — activates → MetaCyc:CPLX-7918; object experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)); After EDTA treatment, 1 mM Cu2+ for 5 min on ice restored 7% of untreated activity. The oxygen-consumption assay used gallate; this is a treatment-specific in vitro effect, not a physiological metal preference. EDTA is normalized to its named parent without assigning the supplied salt or assay microspecies.; Evidence: PMID:16030198activates CHEBI:16318 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Synthetic desB in pMAL-c5x, expressed in E. coli BL21(DE3); anaerobically purified 91.22-kDa MBP fusion with minor cleaved DesB/MBP. Complete originating accession, inserted sequence and fusion junction were not supplied.; Evidence PMID:30346405: fusion construct (PMC6235412 Representative Results paragraph beginning “Protein purification”; Figure 3 fusion material); 2018 4-nitrocatechol inhibition of gallate-dependent oxygen consumption: discussion reports 36.6% at fivefold inhibitor excess and Ki 2.3 ± 0.3 mM. The 1 mM gallate assay tested 0.05–20 mM inhibitor. Figure 7 ratio labels conflict with decreasing activity; no binding-site mechanism is established.; Evidence: PMID:30346405inhibits CHEBI:16918 — inhibits → MetaCyc:CPLX-7918; object experimental construct: Synthetic desB in pMAL-c5x, expressed in E. coli BL21(DE3); anaerobically purified 91.22-kDa MBP fusion with minor cleaved DesB/MBP. Complete originating accession, inserted sequence and fusion junction were not supplied.; Evidence PMID:30346405: fusion construct (PMC6235412 Representative Results paragraph beginning “Protein purification”; Figure 3 fusion material); 2018 high-gallate inhibition is supported by Figure 6/Haldane fitting. Figure axis units and discussion-versus-caption kinetic values conflict; retain the qualitative observation in the tested concentration series without averaging the inconsistent values.; Evidence: PMID:30346405inhibits CHEBI:29033 — activates → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); In the 2016 metal-substitution assay, Fe2+ supported 100 ± 4.6% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.; Evidence: PMID:26867578activates CHEBI:48828 — activates → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); In the 2016 metal-substitution assay, Co2+ supported 79 ± 4.0% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.; Evidence: PMID:26867578activates CHEBI:29035 — activates → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); In the 2016 metal-substitution assay, Mn2+ supported 30 ± 1.9% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.; Evidence: PMID:26867578activates CHEBI:29105 — activates → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); In the 2016 metal-substitution assay, Zn2+ supported 17 ± 0.66% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.; Evidence: PMID:26867578activates CHEBI:29034 — activates → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); In the 2016 metal-substitution assay, Fe3+ supported 14 ± 2.2% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.; Evidence: PMID:26867578activates CHEBI:49786 — activates → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); In the 2016 metal-substitution assay, Ni2+ supported 13 ± 0.33% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.; Evidence: PMID:26867578activates CHEBI:29108 — activates → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); In the 2016 metal-substitution assay, Ca2+ supported 11 ± 1.0% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.; Evidence: PMID:26867578activates CHEBI:18420 — activates → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); In the 2016 metal-substitution assay, Mg2+ supported 9.8 ± 0.66% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.; Evidence: PMID:26867578activates CHEBI:48775 — activates → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); In the 2016 metal-substitution assay, Cd2+ supported 7.4 ± 0.66% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.; Evidence: PMID:26867578activates MetaCyc:CPLX-8178 — has_cofactor → CHEBI:29033; subject experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); 2016 reconstituted WT GalB supports CHM hydration with Fe2+. Table 4 gives a full saturation series. This is an experimentally usable alternative metal in the specified preparation; aerobic KT2442 purification predominantly retained Zn and does not establish exclusive in vivo occupancy.; Evidence: PMID:26867578has cofactor MetaCyc:CPLX-8178 — has_cofactor → CHEBI:48828; subject experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence); 2016 reconstituted WT GalB supports CHM hydration with Co2+. Table 4 gives a full saturation series. This is an experimentally usable alternative metal in the specified preparation; aerobic KT2442 purification predominantly retained Zn and does not establish exclusive in vivo occupancy.; Evidence: PMID:26867578has cofactor CHEBI:4735 — inhibits → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB in the EDTA-present activity row, separate from the washed apo and metal-supplemented rows. Methods specify 50 µM CHM with 50 µM EDTA for the comparison; separate Zn-loaded time courses used 250/1000 µM EDTA. Complete GalB insert/termini remain unresolved; this is not a reconstituted-metal assay.; Evidence PMID:26867578: Table 2 separately lists EDTA, Apo and metal-supplemented GalB activity rows. The EDTA value is 0.030 ± 0.0033%; the Apo value is 4.0 ± 0.33%. (PMC4817193 #T2 EDTA/Apo rows and footnote); Evidence PMID:26867578: EDTA (PMC4817193 #sec16 Enzyme Assays, metal-ion-specificity assay sentence); 2016 EDTA treatment reduces CHM hydratase activity. Table 2 EDTA row is 0.030 ± 0.0033% relative to Fe2+. Separate Zn-loaded time courses gave half-lives 32.3 min at 250 µM EDTA and 17.1 min at 1 mM; 12 h at 1 mM lowered activity below assay error. The 2011 post-treatment rescue failure used a different protocol and is not overwritten. EDTA named-parent normalization does not establish its supplied salt/protonation.; Evidence: PMID:26867578inhibits CHEBI:48828 — activates → MetaCyc:CPLX-8178; object experimental construct: GalB overexpressed in P. putida KT2442 from pVLT31, aerobically purified by three chromatographic steps; the Table 2 native preparation precedes EDTA stripping. The complete GalB insert is unresolved.; Evidence PMID:26867578: Table 2 distinguishes the purified native enzyme before EDTA treatment and its cobalt-supplemented counterpart. (PMC4817193 #T2 Native/Native + Co2+ rows and footnote); In the separately identified KT2442 overexpression/purification preparation, 50 µM CoCl2 for 10 min increased relative CHM activity from 8.6 ± 0.95% to 34 ± 1.5%. This indicates underloading/loss during preparation, not a physiological cobalt preference.; Evidence: PMID:26867578activates NCBIProtein:BAI50711.1 — catalyzes → RHEA:17403; subject experimental construct: Codon-optimized Comamonas E6 pmdE (BAI50711.1) synthesized into pET28a, expressed in E. coli BL21(λDE3), initially N-His6-tagged. Thrombin cleavage and Ni-NTA removal of tag/tagged material were performed; full insert and residual cleavage scar remain unresolved.; Evidence PMID:26867578: polyhistidine tag (PMC4817193 #sec8 Protein Purification, LigJCsE6 tag-removal paragraph); The 2016 Comamonas sp. E6 PmdE/LigJ preparation substitutes for GalB in the GalA–GalD–hydratase–GalC coupled assay, giving 1:1 NADH oxidation relative to added gallate. With GalD omitted, only slow hydratase-independent turnover remains. Polarimetry identifies the same negative CHA enantiomer as GalB, without proving absolute S configuration. The source uses the GalD-generated E,E enol; this exact E6 construct is distinct from the older native PmdE homology annotation and modern SYK-6 keto-KCH reaction.; Evidence: PMID:26867578catalyzes MetaCyc:CPLX-7918 — catalyzes → RHEA:28928; subject experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli; cited 2009 preparation used BL21(DE3) and POROS PI/HQ. Exact tag/vector termini are not supplied. For solution assays, 20-fold EDTA treatment for 12 h at 4 °C preceded anaerobic reactivation with equal volumes of protein and FeCl2/ascorbate solution.; Evidence PMID:24657997: overexpressed using Escherichia coli (PLOS XML #s2a first paragraph; #s2f supplies reactivation context); Reactivated recombinant DesB depleted gallate at 116.1 ± 0.5 U/mg in air-saturated 50 mM Tris-HCl pH 7.5, 30 °C, correcting for product absorbance and substrate autodegradation. This is a different preparation/assay from 2005. The bound-gallate crystal supports substrate coordination but does not identify the immediate solution product tautomer. The shifted iron-site interpretation is a structural model, not directly observed proton transfer.; Evidence: PMID:24657997catalyzes MetaCyc:CPLX-7918 — has_cofactor → CHEBI:29033; subject experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli; cited 2009 preparation used BL21(DE3) and POROS PI/HQ. Exact tag/vector termini are not supplied. For solution assays, 20-fold EDTA treatment for 12 h at 4 °C preceded anaerobic reactivation with equal volumes of protein and FeCl2/ascorbate solution.; Evidence PMID:24657997: overexpressed using Escherichia coli (PLOS XML #s2a first paragraph; #s2f supplies reactivation context); Anaerobic structures place Fe(II) at the intersubunit active sites; solution activity used iron/ascorbate-reconstituted apoenzyme. Static coordination distances and the refined alternative iron positions do not establish a two-iron stoichiometry or a directly observed proton transfer.; Evidence: PMID:24657997has cofactor MetaCyc:CPLX-7918 — has_part → MetaCyc:MONOMER-15119; subject experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli, crystallized anaerobically. Exact tag/vector termini are not supplied; the crystal assembly is distinguished from the 2021 DesB-MBP fusion.; Evidence PMID:24657997: overexpressed using Escherichia coli (PLOS XML #s2a first paragraph; #s2f supplies reactivation context); The 2014 crystal is a swapped homodimer: residues 1–300 of one subunit form a protomer with residues 301–C terminus of the other. Active sites lie at the interface. This is not the separate LigAB alpha/beta assembly and does not prove that an isolated DesB monomer is catalytic.; Evidence: PMID:24657997has part UniProtKB:E8ZB61 — enables → GO:0046943; subject experimental construct: Repaired 449-residue KTGAL GalT expressed from multicopy pIZGalT in the cited whole-cell uptake experiments. This does not identify the inactive KT2440 frameshift or the unsequenced 2023 genomic insert with repaired GalT.; Evidence PMID:21219457: multicopy plasmid pIZGalT (2011 Results GalT, publisher line224); The broad activity label is scoped here to repaired KTGAL GalT: gallate uptake and less efficient protocatechuate uptake in whole cells. The 449-residue allele is distinct from the inactive KT2440 frameshift annotation. Gallate/PCA Vmax values 8.4/3.2 nmol/min/mg and Km 4.3/9.1 µM are reported; the adjacent prose emphasizes pIZGalT whereas Figure 2B caption names GA-induced KTGAL, so the complete kinetic series is not uniformly reassigned to the multicopy construct. Tested 50 µM negatives remain conditional. No proton stoichiometry or direct ligand-binding measurement is inferred.; Evidence: UniProtKB:E8ZB61, PMID:21219457enables UniProtKB:E8ZB61 — located_in → GO:0005886; Plasma-membrane location is an IEA:TreeGrafter annotation; the separate UniProt membrane/multipass comment has ECO:0000305. Neither is described as a directly imaged localization experiment.; Evidence: UniProtKB:E8ZB61located in UniProtKB:Q88JX6 — enables → GO:0015288; Porin activity is inferred from the IEA:InterPro annotation and Opr-family similarity. In the 2011 KTGAL galP disruption, 10 µM gallate uptake fell from 7.4 to 4.2 nmol/min/mg while growth on gallate was unaffected; this is genetic evidence of a low-concentration uptake contribution, not a purified porin flux or binding assay.; Evidence: UniProtKB:Q88JX6, PMID:21219457enables UniProtKB:Q88JX6 — located_in → GO:0016020; Membrane is the issuing GO annotation (IEA:InterPro). An outer-membrane role is inferred from Opr-family similarity; direct localization and purified transport were not established in the primary uptake study.; Evidence: UniProtKB:Q88JX6located in GO:0046943 — part_of → GO:1905039; Ontology-level activity-to-process context. Both terms remain broad; this does not redefine either GO class as gallate-specific.; Evidence: GO:0046943part of CHEBI:75458 — inhibits → GO:1905039; Only the measured gallate uptake instance of this broad transport process is implicated: 30 µM FCCP inhibited whole-cell uptake of 50 µM gallate by KTGAL carrying pIZGalT. This supports proton-motive-force dependence, not direct GalT binding, inhibition of every carboxylate transport process, or a proton stoichiometry. Figure 2C caption gives µM FCCP/CCCP; a PDF text extraction reading mM is rejected.; Evidence: PMID:21219457inhibits CHEBI:3259 — inhibits → GO:1905039; Only the measured gallate uptake instance of this broad transport process is implicated: 20 µM CCCP inhibited whole-cell uptake of 50 µM gallate by KTGAL carrying pIZGalT. This supports proton-motive-force dependence, not direct GalT binding, inhibition of every carboxylate transport process, or a proton stoichiometry. Figure 2C caption gives µM FCCP/CCCP; a PDF text extraction reading mM is rejected.; Evidence: PMID:21219457inhibits CHEBI:42017 — inhibits → GO:1905039; Only the measured gallate uptake instance of this broad transport process is implicated: 1 mM DNP inhibited whole-cell uptake of 50 µM gallate by KTGAL carrying pIZGalT. This supports proton-motive-force dependence, not direct GalT binding, inhibition of every carboxylate transport process, or a proton stoichiometry. Figure 2C caption gives µM FCCP/CCCP; a PDF text extraction reading mM is rejected.; Evidence: PMID:21219457inhibits CHEBI:59051 — inhibits → MetaCyc:CPLX-8178; object experimental construct: 2011 purified GalB from E. coli BL21(DE3)/pETGalB, purified by DEAE and phenyl chromatography. Table 1 reports 244 residues/27.4 kDa; complete insert and termini are unresolved against the 258-residue deposited reference. DEPC assay: 0.5 mM, 7 min, 25 °C.; Evidence MetaCyc:ENZRXN-18373: The activity has purified heterologous-host evidence for CPLX-8178 linked to PMID21219457. (/ptools-xml/Enzymatic-Reaction/evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]); Evidence PMID:21219457: Table 1 lists GalB as a 244-residue, 27.4-kDa protein in the gal cluster. This does not identify the complete recombinant insert with the independently deposited 258-residue reference. (2011 Table1, galB row: protein size and predicted molecular mass columns); Evidence PMID:21219457: pETGalB (2011 Results GalB publisher line274: E. coli BL21(DE3)(pETGalB); Methods GalB expression and purification); Diethyl pyrocarbonate at 0.5 mM for 7 min at 25 °C reduced purified GalB hydratase activity to 15% of untreated. The result suggests an important histidine but does not identify the modified residue or prove a specific proton-transfer mechanism.; Evidence: PMID:21219457inhibits CHEBI:4735 — inhibits → MetaCyc:CPLX-8178; object experimental construct: 2011 E. coli BL21(DE3)/pETGalB cell extract in the EDTA hydratase experiments. Table 1 reports 244 residues/27.4 kDa; complete insert/termini remain unresolved against the 258-residue reference. This extract assay is separate from purified GalB modification and 2016 washed apoenzyme.; Evidence MetaCyc:ENZRXN-18373: The activity has purified heterologous-host evidence for CPLX-8178 linked to PMID21219457. (/ptools-xml/Enzymatic-Reaction/evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]); Evidence PMID:21219457: Table 1 lists GalB as a 244-residue, 27.4-kDa protein in the gal cluster. This does not identify the complete recombinant insert with the independently deposited 258-residue reference. (2011 Table1, galB row: protein size and predicted molecular mass columns); Evidence PMID:21219457: pETGalB (2011 Results GalB publisher line274: E. coli BL21(DE3)(pETGalB); Methods GalB expression and purification); The 2011 experiment reports concentration-dependent inhibition after EDTA treatment (3 h, 4 °C) and no restoration after the tested post-treatment metal additions. This protocol is distinct from the extensively washed/reconstituted 2016 apoenzyme; the two results are not flattened into a universal rescue claim. EDTA is a named-parent normalization with unspecified supplied salt/protonation.; Evidence: PMID:21219457inhibits CHEBI:16918 — regulates → KEGG:ppu:PP_2518; In citrate-grown KT2440, adding 1 mM gallate for 2 h induced the galA RT-PCR signal in the tested aromatic panel. This is indirect whole-cell transcriptional response, not direct gallate–GalR binding or GalA protein activation. The 2005 insertional growth mutant and the later KTGAL repaired transporter strain are separate experimental contexts.; Evidence: PMID:16030014regulates CHEBI:16918 — regulates → KEGG:ppu:PP_2515; The 2011 KTGAL RT-PCR study reports gallate-inducible galBCD and galTAP transcriptional units; citrate-only cultures lacked the tested intergenic products. This edge denotes an indirect environmental transcriptional response at the stated promoter/gene context. Insertions in galB and galT caused downstream polar effects, so individual-gene necessity is not isolated. The gene locus owner is distinct from the repaired KTGAL GalT protein; no direct GalR ligand binding is established.; Evidence: PMID:21219457regulates CHEBI:16918 — regulates → KEGG:ppu:PP_2519; The 2011 KTGAL RT-PCR study reports gallate-inducible galBCD and galTAP transcriptional units; citrate-only cultures lacked the tested intergenic products. This edge denotes an indirect environmental transcriptional response at the stated promoter/gene context. Insertions in galB and galT caused downstream polar effects, so individual-gene necessity is not isolated. The gene locus owner is distinct from the repaired KTGAL GalT protein; no direct GalR ligand binding is established.; Evidence: PMID:21219457regulates CHEBI:29036 — inhibits → MetaCyc:CPLX-8178; object experimental construct: 2016 WT GalB apo preparation after EDTA stripping and washing, incubated with 50 µM Cu2+ before the CHM assay. Host is not assigned per table row; full insert/termini remain unresolved (2011 244 residues versus reference 258). This is distinct from the separately measured EDTA-present preparation.; Evidence PMID:26867578: Table 2 names the Cu2+ and Apo preparations separately. (PMC4817193 #T2 Cu2+/Apo rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, metal-preincubation sentence); Under the 2016 metal-substitution assay, Cu2+ gave 0.19 ± 0.10% CHM hydration activity versus 4.0 ± 0.33% in the apo row, with Fe2+ = 100%. This is lower activity in a 50 µM Cu2+-challenged apo preparation (50 µM CHM, 100 mM HEPES pH 7.0, 25 °C), not a measured physiological inhibition or an untreated Zn-loaded GalB effect. Metal stripping/washing and preincubation conditions are distinct from the EDTA-present row.; Evidence: PMID:26867578inhibits MetaCyc:G1G01-2699-MONOMER — located_in → GO:0005829; The native KT2440 GalB monomer has an explicit cytosol location and a matching GO annotation supported by EV-COMP-AINF with PSORTdb (PMID:26602691). This preserves a computational annotation, not a direct localization assay. It does not assign localization to the separately qualified 2011 or 2016 recombinant preparations or to every pathway component.; Evidence: MetaCyc:G1G01-2699-MONOMERlocated in gallate; CHEBI:16918; Declared type: small_molecule small molecule gallate dioxygen; CHEBI:15379; Declared type: small_molecule small molecule dioxygen (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate; CHEBI:57471; Declared type: small_molecule small molecule (1E)-4-oxobut-1-ene-1,2,4- tricarboxylate hydron; CHEBI:15378; Declared type: small_molecule small molecule hydron 4-carboxy-2-hydroxy-cis,cis-muconate(3-); CHEBI:58142; Declared type: small_molecule small molecule 4-carboxy-2-hydroxy-cis,cis- muconate(3-) water; CHEBI:15377; Declared type: small_molecule small molecule water 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate; CHEBI:58075; Declared type: small_molecule small molecule 2-hydroxy-4-oxobutane-1,2,4- tricarboxylate pyruvate; CHEBI:15361; Declared type: small_molecule small molecule pyruvate oxaloacetate(2-); CHEBI:16452; Declared type: small_molecule small molecule oxaloacetate(2-) protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-2701; Declared type: complex complex protocatechuate 4,5-dioxygenase HMG aldolase; MetaCyc:CPLX-7917; Declared type: complex complex HMG aldolase gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex complex gallate dioxygenase iron(2+); CHEBI:29033; Declared type: cofactor cofactor iron(2+) gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex complex gallate dioxygenase 4-oxalomesaconate tautomerase; MetaCyc:G1G01-2697-MONOMER; Declared type: protein protein 4-oxalomesaconate tautomerase 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex complex 4-oxalmesaconate hydratase zinc(2+); CHEBI:29105; Declared type: cofactor cofactor zinc(2+) 4-oxalomesaconate hydratase; MetaCyc:CPLX-2781; Declared type: complex complex 4-oxalomesaconate hydratase HMG aldolase; MetaCyc:CPLX-2782; Declared type: complex complex HMG aldolase magnesium(2+); CHEBI:18420; Declared type: cofactor cofactor magnesium(2+) 4-carboxy-4-hydroxy-2-oxoadipate aldolase; MetaCyc:CPLX-2865; Declared type: complex complex 4-carboxy-4-hydroxy-2- oxoadipate aldolase HMG aldolase; MetaCyc:CPLX-2881; Declared type: complex complex HMG aldolase manganese(2+); CHEBI:29035; Declared type: cofactor cofactor manganese(2+) 4-hydroxy-4-methyl-2-oxoglutarate aldolase; MetaCyc:CPLX1G01-155; Declared type: complex complex 4-hydroxy-4-methyl-2- oxoglutarate aldolase protocatechuate 4,5-dioxygenase α subunit; MetaCyc:MONOMER-3165; Declared type: protein protein protocatechuate 4,5-dioxygenase α subunit protocatechuate 4,5-dioxygenase β subunit; MetaCyc:MONOMER-3166; Declared type: protein protein protocatechuate 4,5-dioxygenase β subunit subunit of 4-oxalomesaconate hydratase; MetaCyc:MONOMER-3243; Declared type: protein protein subunit of 4-oxalomesaconate hydratase 4-oxalocitramalate aldolase subunit; MetaCyc:MONOMER-3244; Declared type: protein protein 4-oxalocitramalate aldolase subunit LigK; MetaCyc:MONOMER-3469; Declared type: protein protein LigK subunit of 4-hydroxy-4-methyl-2-oxoglutarate aldolase; MetaCyc:MONOMER-3481; Declared type: protein protein subunit of 4-hydroxy-4- methyl-2-oxoglutarate aldolase HMG aldolase monomer; MetaCyc:MONOMER-3184; Declared type: protein protein HMG aldolase monomer gallate dioxygenase monomer; MetaCyc:MONOMER-15119; Declared type: protein protein gallate dioxygenase monomer gallate dioxygenase monomer; MetaCyc:G1G01-2702-MONOMER; Declared type: protein protein gallate dioxygenase monomer 4-oxalmesaconate hydratase monomer; MetaCyc:G1G01-2699-MONOMER; Declared type: protein protein 4-oxalmesaconate hydratase monomer 4-hydroxy-4-methyl-2-oxoglutarate aldolase aldolase; MetaCyc:G1G01-2698-MONOMER; Declared type: protein protein 4-hydroxy-4-methyl-2- oxoglutarate aldolase aldolase N-ethylmaleimide; MetaCyc:N-ETHYLMALEIMIDE; Declared type: small_molecule small molecule N-ethylmaleimide mercury(2+); CHEBI:16793; Declared type: small_molecule small molecule mercury(2+) p-chloromercuribenzoate; MetaCyc:CPD-29; Declared type: small_molecule small molecule p-chloromercuribenzoate copper sulfate; MetaCyc:CUSO4; Declared type: small_molecule small molecule copper sulfate Gallate transporter; UniProtKB:E8ZB61; Declared type: protein protein Gallate transporter Porin-like protein GalP; UniProtKB:Q88JX6; Declared type: protein protein Porin-like protein GalP HTH-type transcriptional regulator GalR; UniProtKB:Q88JX7; Declared type: protein protein HTH-type transcriptional regulator GalR PP_2515; KEGG:ppu:PP_2515; Declared type: gene gene PP_2515 PP_2519; KEGG:ppu:PP_2519; Declared type: gene gene PP_2519 protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex complex protocatechuate 4,5-dioxygenase protocatechuate 4,5-dioxygenase alpha chain; MetaCyc:MONOMER-15116; Declared type: protein protein protocatechuate 4,5-dioxygenase alpha chain protocatechuate 4,5-dioxygenase beta chain; MetaCyc:MONOMER-15117; Declared type: protein protein protocatechuate 4,5-dioxygenase beta chain 3-O-methylgallate 3,4-dioxygenase; MetaCyc:MONOMER-15115; Declared type: protein protein 3-O-methylgallate 3,4-dioxygenase ethylenediaminetetraacetic acid; CHEBI:4735; Declared type: small_molecule small molecule ethylenediaminetetraacetic acid 2,2'-bipyridine; CHEBI:30351; Declared type: small_molecule small molecule 2,2'-bipyridine hydrogen peroxide; CHEBI:16240; Declared type: small_molecule small molecule hydrogen peroxide copper(2+); CHEBI:29036; Declared type: small_molecule small molecule copper(2+) cobalt(2+); CHEBI:48828; Declared type: cofactor cofactor cobalt(2+) sodium ascorbate; CHEBI:113451; Declared type: small_molecule small molecule sodium ascorbate 3,4-dihydroxybenzoate; CHEBI:36241; Declared type: small_molecule small molecule 3,4-dihydroxybenzoate 4-nitrocatechol; CHEBI:16318; Declared type: small_molecule small molecule 4-nitrocatechol vanillate; CHEBI:16632; Declared type: small_molecule small molecule vanillate 2-Methoxy-4-methylphenol; CHEBI:89886; Declared type: small_molecule small molecule 2-Methoxy-4-methylphenol syringate; CHEBI:132111; Declared type: small_molecule small molecule syringate vanillin; CHEBI:18346; Declared type: small_molecule small molecule vanillin syringaldehyde; CHEBI:67380; Declared type: small_molecule small molecule syringaldehyde 2,3,4-trihydroxybenzoic acid; CHEBI:88714; Declared type: small_molecule small molecule 2,3,4-trihydroxybenzoic acid 2,3-dihydroxybenzoic acid; CHEBI:18026; Declared type: small_molecule small molecule 2,3-dihydroxybenzoic acid 4-methylcatechol; CHEBI:17254; Declared type: small_molecule small molecule 4-methylcatechol 3-methoxycatechol; CHEBI:141700; Declared type: small_molecule small molecule 3-methoxycatechol iron(3+); CHEBI:29034; Declared type: cofactor cofactor iron(3+) nickel(2+); CHEBI:49786; Declared type: cofactor cofactor nickel(2+) calcium(2+); CHEBI:29108; Declared type: cofactor cofactor calcium(2+) cadmium(2+); CHEBI:48775; Declared type: cofactor cofactor cadmium(2+) 4-oxalomesaconate hydratase [Comamonas sp. E6]; NCBIProtein:BAI50711.1; Declared type: protein protein 4-oxalomesaconate hydratase [Comamonas sp. E6] carbonyl cyanide p-trifluoromethoxyphenylhydrazone; CHEBI:75458; Declared type: small_molecule small molecule carbonyl cyanide p- trifluoromethoxyphenylhydraz one CCCP; CHEBI:3259; Declared type: small_molecule small molecule CCCP 2,4-dinitrophenol; CHEBI:42017; Declared type: small_molecule small molecule 2,4-dinitrophenol diethyl pyrocarbonate; CHEBI:59051; Declared type: small_molecule small molecule diethyl pyrocarbonate plasma membrane; GO:0005886; Declared type: cellular_component cellular component plasma membrane membrane; GO:0016020; Declared type: cellular_component cellular component membrane PP_2518; KEGG:ppu:PP_2518; Declared type: gene gene PP_2518 cytosol; GO:0005829; Declared type: cellular_component cellular component cytosol 3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+); RHEA:28928; Declared type: molecular_activity; Source reaction direction: left_to_right molecular activity 3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene- 1,2,4-tricarboxylate + 2 H(+) (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate => 4-carboxy-2-hydroxy-cis,cis-muconate; RHEA:28932; Declared type: molecular_activity; Source reaction direction: reversible molecular activity (1E)-4-oxobut-1-ene-1,2,4- tricarboxylate => 4-carboxy-2-hydroxy-cis,cis- muconate 4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate; RHEA:17403; Declared type: molecular_activity; Source reaction direction: reversible molecular activity 4-carboxy-2-hydroxy-cis,cis- muconate + H2O => 2-hydroxy- 4-oxobutane-1,2,4- tricarboxylate 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate; RHEA:28936; Declared type: molecular_activity; Source reaction direction: reversible molecular activity 2-hydroxy-4-oxobutane-1,2,4- tricarboxylate => oxaloacetate + pyruvate TCA cycle I (prokaryotic); MetaCyc:TCA; Declared type: pathway pathway TCA cycle I (prokaryotic) syringate degradation; MetaCyc:PWY-6339; Declared type: pathway pathway syringate degradation carboxylic acid transmembrane transporter activity; GO:0046943; Declared type: molecular_activity molecular activity carboxylic acid transmembrane transporter activity carboxylic acid transmembrane transport; GO:1905039; Declared type: biological_process biological process carboxylic acid transmembrane transport porin activity; GO:0015288; Declared type: molecular_activity molecular activity porin activity gallate degradation I; MetaCyc:GALLATE-DEGRADATION-II-PWY; Declared type: pathway pathway gallate degradation I gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) in E. coli BL21(DE3), purified by POROS chromatography; initiating methionine processed. Full terminal construct sequence and any vector-derived residues were not established.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)); Evidence PMID:16030198: appeared to be processed (PMC1196043 Results, Purification of DesB, first paragraph, initiating methionine sentence following the N-terminal sequence) experimental construct gallate dioxygenase [experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) in E. coli BL21(DE3), purified by POROS chromatography;…] 4-oxalomesaconate tautomerase; MetaCyc:G1G01-2697-MONOMER; Declared type: protein; experimental construct: 2011 KT2440 GalD expressed from pETGalDc in E. coli BL21(DE3), assayed in partly purified extract. This is separate from the explicitly His6-retaining 2016 GalD preparation.; Evidence PMID:21219457: pETGalDc (2011 publisher Figure 3 caption line 249 and Results line 253; Methods lines 349, 365–366 and 376–377 (construct, BL21(DE3) expression, extract preparation). Retained main-text transcription; original spectra and Table S1 were inaccessible.) experimental construct 4-oxalomesaconate tautomerase [experimental construct: 2011 KT2440 GalD expressed from pETGalDc in E. coli BL21(DE3), assayed in partly purified extract.…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2011 pET29a-derived KT2440 GalB in E. coli BL21(DE3), purified by DEAE and phenyl chromatography. Table 1 reports 244 residues; reference Q88JX8 has 258. Complete insert/termini remain unresolved; the separate 2016 crystal is 240 residues.; Evidence MetaCyc:ENZRXN-18373: The activity has purified heterologous-host evidence for CPLX-8178 linked to PMID21219457. (/ptools-xml/Enzymatic-Reaction/evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]); Evidence PMID:21219457: Table 1 lists GalB as a 244-residue, 27.4-kDa protein in the gal cluster. This does not identify the complete recombinant insert with the independently deposited 258-residue reference. (2011 Table1, galB row: protein size and predicted molecular mass columns); Evidence PMID:21219457: pETGalB (2011 Results GalB publisher line274: E. coli BL21(DE3)(pETGalB); Methods GalB expression and purification) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2011 pET29a-derived KT2440 GalB in E. coli BL21(DE3), purified by DEAE and phenyl chromatography.…] 4-hydroxy-4-methyl-2-oxoglutarate aldolase; MetaCyc:CPLX1G01-155; Declared type: complex; experimental construct: 2011 KT2440 GalC expressed in E. coli BL21(DE3) from pETGalC, assayed as crude extract. Historical purified GalC/hexamer annotations do not establish the sequence or assembly of this recombinant preparation.; Evidence PMID:21219457: pETGalC (2011 publisher Results lines 307–309 and Methods lines 349, 365–366 and 380 (construct, BL21(DE3), crude extract, assay and activities). Retained main-text transcription; original Table S1 and Figure S7 were inaccessible.) experimental construct 4-hydroxy-4-methyl-2- oxoglutarate aldolase [experimental construct: 2011 KT2440 GalC expressed in E. coli BL21(DE3) from pETGalC, assayed as crude extract. Historical…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established. The encoded chain is 420 aa; Edman results suggest 419 aa after initiator-Met processing.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).); Evidence NCBIProtein:AAN68130.2: The PP_2518 CDS names protein AAN68130.2 and supplies a 420-residue translation beginning MARIIGG. (AE015451.2 CDS complement(2865514..2866776), /locus_tag, /protein_id and /translation.); Evidence PMID:16030014: appeared to be processed. (Results cloning/expression, p.35385; gal2005_methods-successor-web.json L1011-L1019, exact phrase at L1015.) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] gallate dioxygenase; MetaCyc:CPLX-8177; Declared type: complex; experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.; Evidence PMID:16030014: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg. (Table 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).) experimental construct gallate dioxygenase [experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and…] HTH-type transcriptional regulator GalR; UniProtKB:Q88JX7; Declared type: protein; experimental construct: GalR supplied in an additional plasmid copy in P. putida KT2440, with PP_RS13150-RFP on pIK002_T; the promoter-only pIK002A_T control retains endogenous GalR. Readout: 6 h after 1.25 mM gallate in LB/MM. Native target-gene transcription and direct GalR binding were not measured.; Evidence PMID:36724292: extra copy of galR gene (PMC9942251 XML body paragraph ordinal 10 (zero-based 9); experimental comparison in body paragraph ordinal 8 (zero-based 7) and Figure 2B.) experimental construct HTH-type transcriptional regulator GalR [experimental construct: GalR supplied in an additional plasmid copy in P. putida KT2440, with PP_RS13150-RFP on pIK002_T;…] HTH-type transcriptional regulator GalR; UniProtKB:Q88JX7; Declared type: protein; experimental construct: GalR supplied in an additional plasmid copy in P. putida KT2440, with PP_RS13170-RFP on pIK014_T; the promoter-only pIK014A_T control retains endogenous GalR. Readout: 6 h after 1.25 mM gallate in LB/MM. Native target-gene transcription and direct GalR binding were not measured.; Evidence PMID:36724292: extra copy of galR gene (PMC9942251 XML body paragraph ordinal 10 (zero-based 9); experimental comparison in body paragraph ordinal 8 (zero-based 7) and Figure 2B.) experimental construct HTH-type transcriptional regulator GalR [experimental construct: GalR supplied in an additional plasmid copy in P. putida KT2440, with PP_RS13170-RFP on pIK014_T;…] protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex; experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification) experimental construct protocatechuate 4,5-dioxygenase [experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not…] protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex; experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification) experimental construct protocatechuate 4,5-dioxygenase [experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not…] protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex; experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification) experimental construct protocatechuate 4,5-dioxygenase [experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not…] protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex; experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification) experimental construct protocatechuate 4,5-dioxygenase [experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not…] protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex; experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification) experimental construct protocatechuate 4,5-dioxygenase [experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not…] protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex; experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification) experimental construct protocatechuate 4,5-dioxygenase [experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not…] protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex; experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification) experimental construct protocatechuate 4,5-dioxygenase [experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not…] protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex; experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification) experimental construct protocatechuate 4,5-dioxygenase [experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not…] protocatechuate 4,5-dioxygenase; MetaCyc:CPLX-7916; Declared type: complex; experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.; Evidence PMID:23977959: His-tag cleaved (#S4/#P16; retained original HTML line1422, before Source-Q purification) experimental construct protocatechuate 4,5-dioxygenase [experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)) experimental construct gallate dioxygenase [experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)) experimental construct gallate dioxygenase [experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)) experimental construct gallate dioxygenase [experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)) experimental construct gallate dioxygenase [experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)) experimental construct gallate dioxygenase [experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)) experimental construct gallate dioxygenase [experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused protein (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)) experimental construct gallate dioxygenase [experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.; Evidence DOI:10.1016/j.procbio.2021.01.019: DesB-MBP fused (Results Anaerobic Purification, author-deposited maintext line913 (desb2021_rg_mid-web.json)) experimental construct gallate dioxygenase [experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)) experimental construct gallate dioxygenase [experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns.…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)) experimental construct gallate dioxygenase [experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns.…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)) experimental construct gallate dioxygenase [experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns.…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.; Evidence PMID:16030198: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host. (PMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts); Evidence PMID:16030198: cells harboring pETB (PMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)) experimental construct gallate dioxygenase [experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns.…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Synthetic desB in pMAL-c5x, expressed in E. coli BL21(DE3); anaerobically purified 91.22-kDa MBP fusion with minor cleaved DesB/MBP. Complete originating accession, inserted sequence and fusion junction were not supplied.; Evidence PMID:30346405: fusion construct (PMC6235412 Representative Results paragraph beginning “Protein purification”; Figure 3 fusion material) experimental construct gallate dioxygenase [experimental construct: Synthetic desB in pMAL-c5x, expressed in E. coli BL21(DE3); anaerobically purified 91.22-kDa MBP…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: Synthetic desB in pMAL-c5x, expressed in E. coli BL21(DE3); anaerobically purified 91.22-kDa MBP fusion with minor cleaved DesB/MBP. Complete originating accession, inserted sequence and fusion junction were not supplied.; Evidence PMID:30346405: fusion construct (PMC6235412 Representative Results paragraph beginning “Protein purification”; Figure 3 fusion material) experimental construct gallate dioxygenase [experimental construct: Synthetic desB in pMAL-c5x, expressed in E. coli BL21(DE3); anaerobically purified 91.22-kDa MBP…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.; Evidence PMID:26867578: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type. (PMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31;…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB in the EDTA-present activity row, separate from the washed apo and metal-supplemented rows. Methods specify 50 µM CHM with 50 µM EDTA for the comparison; separate Zn-loaded time courses used 250/1000 µM EDTA. Complete GalB insert/termini remain unresolved; this is not a reconstituted-metal assay.; Evidence PMID:26867578: Table 2 separately lists EDTA, Apo and metal-supplemented GalB activity rows. The EDTA value is 0.030 ± 0.0033%; the Apo value is 4.0 ± 0.33%. (PMC4817193 #T2 EDTA/Apo rows and footnote); Evidence PMID:26867578: EDTA (PMC4817193 #sec16 Enzyme Assays, metal-ion-specificity assay sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB in the EDTA- present activity row, separate from the washed apo and metal-supplemented…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: GalB overexpressed in P. putida KT2442 from pVLT31, aerobically purified by three chromatographic steps; the Table 2 native preparation precedes EDTA stripping. The complete GalB insert is unresolved.; Evidence PMID:26867578: Table 2 distinguishes the purified native enzyme before EDTA treatment and its cobalt-supplemented counterpart. (PMC4817193 #T2 Native/Native + Co2+ rows and footnote) experimental construct 4-oxalmesaconate hydratase [experimental construct: GalB overexpressed in P. putida KT2442 from pVLT31, aerobically purified by three chromatographic…] 4-oxalomesaconate hydratase [Comamonas sp. E6]; NCBIProtein:BAI50711.1; Declared type: protein; experimental construct: Codon-optimized Comamonas E6 pmdE (BAI50711.1) synthesized into pET28a, expressed in E. coli BL21(λDE3), initially N-His6-tagged. Thrombin cleavage and Ni-NTA removal of tag/tagged material were performed; full insert and residual cleavage scar remain unresolved.; Evidence PMID:26867578: polyhistidine tag (PMC4817193 #sec8 Protein Purification, LigJCsE6 tag-removal paragraph) experimental construct 4-oxalomesaconate hydratase [Comamonas sp. E6] [experimental construct: Codon-optimized Comamonas E6 pmdE (BAI50711.1) synthesized into pET28a, expressed in E. coli…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli; cited 2009 preparation used BL21(DE3) and POROS PI/HQ. Exact tag/vector termini are not supplied. For solution assays, 20-fold EDTA treatment for 12 h at 4 °C preceded anaerobic reactivation with equal volumes of protein and FeCl2/ascorbate solution.; Evidence PMID:24657997: overexpressed using Escherichia coli (PLOS XML #s2a first paragraph; #s2f supplies reactivation context) experimental construct gallate dioxygenase [experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli; cited 2009 preparation used BL21(DE3) and…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli; cited 2009 preparation used BL21(DE3) and POROS PI/HQ. Exact tag/vector termini are not supplied. For solution assays, 20-fold EDTA treatment for 12 h at 4 °C preceded anaerobic reactivation with equal volumes of protein and FeCl2/ascorbate solution.; Evidence PMID:24657997: overexpressed using Escherichia coli (PLOS XML #s2a first paragraph; #s2f supplies reactivation context) experimental construct gallate dioxygenase [experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli; cited 2009 preparation used BL21(DE3) and…] gallate dioxygenase; MetaCyc:CPLX-7918; Declared type: complex; experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli, crystallized anaerobically. Exact tag/vector termini are not supplied; the crystal assembly is distinguished from the 2021 DesB-MBP fusion.; Evidence PMID:24657997: overexpressed using Escherichia coli (PLOS XML #s2a first paragraph; #s2f supplies reactivation context) experimental construct gallate dioxygenase [experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli, crystallized anaerobically. Exact tag/vector…] Gallate transporter; UniProtKB:E8ZB61; Declared type: protein; experimental construct: Repaired 449-residue KTGAL GalT expressed from multicopy pIZGalT in the cited whole-cell uptake experiments. This does not identify the inactive KT2440 frameshift or the unsequenced 2023 genomic insert with repaired GalT.; Evidence PMID:21219457: multicopy plasmid pIZGalT (2011 Results GalT, publisher line224) experimental construct Gallate transporter [experimental construct: Repaired 449-residue KTGAL GalT expressed from multicopy pIZGalT in the cited whole-cell uptake…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2011 purified GalB from E. coli BL21(DE3)/pETGalB, purified by DEAE and phenyl chromatography. Table 1 reports 244 residues/27.4 kDa; complete insert and termini are unresolved against the 258-residue deposited reference. DEPC assay: 0.5 mM, 7 min, 25 °C.; Evidence MetaCyc:ENZRXN-18373: The activity has purified heterologous-host evidence for CPLX-8178 linked to PMID21219457. (/ptools-xml/Enzymatic-Reaction/evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]); Evidence PMID:21219457: Table 1 lists GalB as a 244-residue, 27.4-kDa protein in the gal cluster. This does not identify the complete recombinant insert with the independently deposited 258-residue reference. (2011 Table1, galB row: protein size and predicted molecular mass columns); Evidence PMID:21219457: pETGalB (2011 Results GalB publisher line274: E. coli BL21(DE3)(pETGalB); Methods GalB expression and purification) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2011 purified GalB from E. coli BL21(DE3)/pETGalB, purified by DEAE and phenyl chromatography.…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2011 E. coli BL21(DE3)/pETGalB cell extract in the EDTA hydratase experiments. Table 1 reports 244 residues/27.4 kDa; complete insert/termini remain unresolved against the 258-residue reference. This extract assay is separate from purified GalB modification and 2016 washed apoenzyme.; Evidence MetaCyc:ENZRXN-18373: The activity has purified heterologous-host evidence for CPLX-8178 linked to PMID21219457. (/ptools-xml/Enzymatic-Reaction/evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]); Evidence PMID:21219457: Table 1 lists GalB as a 244-residue, 27.4-kDa protein in the gal cluster. This does not identify the complete recombinant insert with the independently deposited 258-residue reference. (2011 Table1, galB row: protein size and predicted molecular mass columns); Evidence PMID:21219457: pETGalB (2011 Results GalB publisher line274: E. coli BL21(DE3)(pETGalB); Methods GalB expression and purification) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2011 E. coli BL21(DE3)/pETGalB cell extract in the EDTA hydratase experiments. Table 1 reports 244…] 4-oxalmesaconate hydratase; MetaCyc:CPLX-8178; Declared type: complex; experimental construct: 2016 WT GalB apo preparation after EDTA stripping and washing, incubated with 50 µM Cu2+ before the CHM assay. Host is not assigned per table row; full insert/termini remain unresolved (2011 244 residues versus reference 258). This is distinct from the separately measured EDTA-present preparation.; Evidence PMID:26867578: Table 2 names the Cu2+ and Apo preparations separately. (PMC4817193 #T2 Cu2+/Apo rows); Evidence PMID:26867578: apoGalB (PMC4817193 #sec16 Enzyme Assays, metal-preincubation sentence) experimental construct 4-oxalmesaconate hydratase [experimental construct: 2016 WT GalB apo preparation after EDTA stripping and washing, incubated with 50 µM Cu2+ before the…]

Components and activities

Declared biological components and source reaction directions
NodeKind or roleSource direction
gallate
CHEBI:16918		small molecule
dioxygen
CHEBI:15379		small molecule
(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate
CHEBI:57471		small molecule
hydron
CHEBI:15378		small molecule
4-carboxy-2-hydroxy-cis,cis-muconate(3-)
CHEBI:58142		small molecule
water
CHEBI:15377		small molecule
2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
CHEBI:58075		small molecule
pyruvate
CHEBI:15361		small molecule
oxaloacetate(2-)
CHEBI:16452		small molecule
protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-2701		complex
HMG aldolase
MetaCyc:CPLX-7917		complex
gallate dioxygenase
MetaCyc:CPLX-7918		complex
iron(2+)
CHEBI:29033		cofactor
gallate dioxygenase
MetaCyc:CPLX-8177		complex
4-oxalomesaconate tautomerase
MetaCyc:G1G01-2697-MONOMER		protein
4-oxalmesaconate hydratase
MetaCyc:CPLX-8178		complex
zinc(2+)
CHEBI:29105		cofactor
4-oxalomesaconate hydratase
MetaCyc:CPLX-2781		complex
HMG aldolase
MetaCyc:CPLX-2782		complex
magnesium(2+)
CHEBI:18420		cofactor
4-carboxy-4-hydroxy-2-oxoadipate aldolase
MetaCyc:CPLX-2865		complex
HMG aldolase
MetaCyc:CPLX-2881		complex
manganese(2+)
CHEBI:29035		cofactor
4-hydroxy-4-methyl-2-oxoglutarate aldolase
MetaCyc:CPLX1G01-155		complex
protocatechuate 4,5-dioxygenase α subunit
MetaCyc:MONOMER-3165		protein
protocatechuate 4,5-dioxygenase β subunit
MetaCyc:MONOMER-3166		protein
subunit of 4-oxalomesaconate hydratase
MetaCyc:MONOMER-3243		protein
4-oxalocitramalate aldolase subunit
MetaCyc:MONOMER-3244		protein
LigK
MetaCyc:MONOMER-3469		protein
subunit of 4-hydroxy-4-methyl-2-oxoglutarate aldolase
MetaCyc:MONOMER-3481		protein
HMG aldolase monomer
MetaCyc:MONOMER-3184		protein
gallate dioxygenase monomer
MetaCyc:MONOMER-15119		protein
gallate dioxygenase monomer
MetaCyc:G1G01-2702-MONOMER		protein
4-oxalmesaconate hydratase monomer
MetaCyc:G1G01-2699-MONOMER		protein
4-hydroxy-4-methyl-2-oxoglutarate aldolase aldolase
MetaCyc:G1G01-2698-MONOMER		protein
N-ethylmaleimide
MetaCyc:N-ETHYLMALEIMIDE		small molecule
mercury(2+)
CHEBI:16793		small molecule
p-chloromercuribenzoate
MetaCyc:CPD-29		small molecule
copper sulfate
MetaCyc:CUSO4		small molecule
Gallate transporter
UniProtKB:E8ZB61		protein
Porin-like protein GalP
UniProtKB:Q88JX6		protein
HTH-type transcriptional regulator GalR
UniProtKB:Q88JX7		protein
PP_2515
KEGG:ppu:PP_2515		gene
PP_2519
KEGG:ppu:PP_2519		gene
protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916		complex
protocatechuate 4,5-dioxygenase alpha chain
MetaCyc:MONOMER-15116		protein
protocatechuate 4,5-dioxygenase beta chain
MetaCyc:MONOMER-15117		protein
3-O-methylgallate 3,4-dioxygenase
MetaCyc:MONOMER-15115		protein
ethylenediaminetetraacetic acid
CHEBI:4735		small molecule
2,2'-bipyridine
CHEBI:30351		small molecule
hydrogen peroxide
CHEBI:16240		small molecule
copper(2+)
CHEBI:29036		small molecule
cobalt(2+)
CHEBI:48828		cofactor
sodium ascorbate
CHEBI:113451		small molecule
3,4-dihydroxybenzoate
CHEBI:36241		small molecule
4-nitrocatechol
CHEBI:16318		small molecule
vanillate
CHEBI:16632		small molecule
2-Methoxy-4-methylphenol
CHEBI:89886		small molecule
syringate
CHEBI:132111		small molecule
vanillin
CHEBI:18346		small molecule
syringaldehyde
CHEBI:67380		small molecule
2,3,4-trihydroxybenzoic acid
CHEBI:88714		small molecule
2,3-dihydroxybenzoic acid
CHEBI:18026		small molecule
4-methylcatechol
CHEBI:17254		small molecule
3-methoxycatechol
CHEBI:141700		small molecule
iron(3+)
CHEBI:29034		cofactor
nickel(2+)
CHEBI:49786		cofactor
calcium(2+)
CHEBI:29108		cofactor
cadmium(2+)
CHEBI:48775		cofactor
4-oxalomesaconate hydratase [Comamonas sp. E6]
NCBIProtein:BAI50711.1		protein
carbonyl cyanide p-trifluoromethoxyphenylhydrazone
CHEBI:75458		small molecule
CCCP
CHEBI:3259		small molecule
2,4-dinitrophenol
CHEBI:42017		small molecule
diethyl pyrocarbonate
CHEBI:59051		small molecule
plasma membrane
GO:0005886		cellular component
membrane
GO:0016020		cellular component
PP_2518
KEGG:ppu:PP_2518		gene
cytosol
GO:0005829		cellular component
3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928		molecular activityleft to right
(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate => 4-carboxy-2-hydroxy-cis,cis-muconate
RHEA:28932		molecular activityreversible
4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403		molecular activityreversible
2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936		molecular activityreversible
TCA cycle I (prokaryotic)
MetaCyc:TCA		pathway
syringate degradation
MetaCyc:PWY-6339		pathway
carboxylic acid transmembrane transporter activity
GO:0046943		molecular activity
carboxylic acid transmembrane transport
GO:1905039		biological process
porin activity
GO:0015288		molecular activity

Mechanistic edges

Mechanistic edges and their cited evidence
SubjectPredicate and descriptionObjectEvidence
3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928		part_of

Formal native membership; accession suffix II is distinct from displayed name I.

MetaCyc:GALLATE-DEGRADATION-II-PWY
  • MetaCyc:GALLATE-DEGRADATION-II-PWY

    Source assertion: GALLATE-DIOXYGENASE-RXN is one of the four formal reaction-list members of gallate degradation I.

    Source location/ptools-xml/Pathway/reaction-list/Reaction[@frameid='GALLATE-DIOXYGENASE-RXN']
3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928		has_input

Selected physiological route orientation; native reversible metadata retained independently.

gallate
CHEBI:16918
  • MetaCyc:GALLATE-DIOXYGENASE-RXN

    Source assertion: Selected L2R pathway flux gives 1 CPD-183 on the substrates side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='GALLATE-DIOXYGENASE-RXN']/left[Compound/@frameid='CPD-183']
3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928		has_input

Selected physiological route orientation; native reversible metadata retained independently.

dioxygen
CHEBI:15379
  • MetaCyc:GALLATE-DIOXYGENASE-RXN

    Source assertion: Selected L2R pathway flux gives 1 OXYGEN-MOLECULE on the substrates side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='GALLATE-DIOXYGENASE-RXN']/left[Compound/@frameid='OXYGEN-MOLECULE']
3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928		has_output

Selected physiological route orientation; native reversible metadata retained independently.

(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate
CHEBI:57471
  • MetaCyc:GALLATE-DIOXYGENASE-RXN

    Source assertion: Selected L2R pathway flux gives 1 4-OXALOMESACONATE on the products side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='GALLATE-DIOXYGENASE-RXN']/right[Compound/@frameid='4-OXALOMESACONATE']
3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928		has_output

Selected physiological route orientation; native reversible metadata retained independently.

hydron
CHEBI:15378
  • MetaCyc:GALLATE-DIOXYGENASE-RXN

    Source assertion: Selected L2R pathway flux gives 2 PROTON on the products side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='GALLATE-DIOXYGENASE-RXN']/right[Compound/@frameid='PROTON']
(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate => 4-carboxy-2-hydroxy-cis,cis-muconate
RHEA:28932		part_of

Formal native membership; accession suffix II is distinct from displayed name I.

MetaCyc:GALLATE-DEGRADATION-II-PWY
  • MetaCyc:GALLATE-DEGRADATION-II-PWY

    Source assertion: RXN-9983 is one of the four formal reaction-list members of gallate degradation I.

    Source location/ptools-xml/Pathway/reaction-list/Reaction[@frameid='RXN-9983']
(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate => 4-carboxy-2-hydroxy-cis,cis-muconate
RHEA:28932		has_input

Selected physiological route orientation; native reversible metadata retained independently.

(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate
CHEBI:57471
  • MetaCyc:RXN-9983

    Source assertion: Selected L2R pathway flux gives 1 4-OXALOMESACONATE on the substrates side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='RXN-9983']/left[Compound/@frameid='4-OXALOMESACONATE']
(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate => 4-carboxy-2-hydroxy-cis,cis-muconate
RHEA:28932		has_output

Selected physiological route orientation; native reversible metadata retained independently.

4-carboxy-2-hydroxy-cis,cis-muconate(3-)
CHEBI:58142
  • MetaCyc:RXN-9983

    Source assertion: Selected L2R pathway flux gives 1 CPD-12849 on the products side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='RXN-9983']/right[Compound/@frameid='CPD-12849']
4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403		part_of

Formal native membership; accession suffix II is distinct from displayed name I.

MetaCyc:GALLATE-DEGRADATION-II-PWY
  • MetaCyc:GALLATE-DEGRADATION-II-PWY

    Source assertion: RXN-2463 is one of the four formal reaction-list members of gallate degradation I.

    Source location/ptools-xml/Pathway/reaction-list/Reaction[@frameid='RXN-2463']
4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403		has_input

Selected physiological route orientation; native reversible metadata retained independently.

4-carboxy-2-hydroxy-cis,cis-muconate(3-)
CHEBI:58142
  • MetaCyc:RXN-2463

    Source assertion: Selected R2L pathway flux gives 1 CPD-12849 on the substrates side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='RXN-2463']/right[Compound/@frameid='CPD-12849']
4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403		has_input

Selected physiological route orientation; native reversible metadata retained independently.

water
CHEBI:15377
  • MetaCyc:RXN-2463

    Source assertion: Selected R2L pathway flux gives 1 WATER on the substrates side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='RXN-2463']/right[Compound/@frameid='WATER']
4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403		has_output

Selected physiological route orientation; native reversible metadata retained independently.

2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
CHEBI:58075
  • MetaCyc:RXN-2463

    Source assertion: Selected R2L pathway flux gives 1 4-OH-4-ACETYL-2-OXOGLUTARATE on the products side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='RXN-2463']/left[Compound/@frameid='4-OH-4-ACETYL-2-OXOGLUTARATE']
2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936		part_of

Formal native membership; accession suffix II is distinct from displayed name I.

MetaCyc:GALLATE-DEGRADATION-II-PWY
  • MetaCyc:GALLATE-DEGRADATION-II-PWY

    Source assertion: RXN-2464 is one of the four formal reaction-list members of gallate degradation I.

    Source location/ptools-xml/Pathway/reaction-list/Reaction[@frameid='RXN-2464']
2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936		has_input

Selected physiological route orientation; native reversible metadata retained independently.

2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
CHEBI:58075
  • MetaCyc:RXN-2464

    Source assertion: Selected L2R pathway flux gives 1 4-OH-4-ACETYL-2-OXOGLUTARATE on the substrates side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='RXN-2464']/left[Compound/@frameid='4-OH-4-ACETYL-2-OXOGLUTARATE']
2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936		has_output

Selected physiological route orientation; native reversible metadata retained independently.

pyruvate
CHEBI:15361
  • MetaCyc:RXN-2464

    Source assertion: Selected L2R pathway flux gives 1 PYRUVATE on the products side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='RXN-2464']/right[Compound/@frameid='PYRUVATE']
2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936		has_output

Selected physiological route orientation; native reversible metadata retained independently.

oxaloacetate(2-)
CHEBI:16452
  • MetaCyc:RXN-2464

    Source assertion: Selected L2R pathway flux gives 1 OXALACETIC_ACID on the products side; complete native equation checked for atoms and charge.

    Source location/ptools-xml/Reaction[@frameid='RXN-2464']/right[Compound/@frameid='OXALACETIC_ACID']
3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928		provides_input_for

Native order and direct material continuity.

(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate => 4-carboxy-2-hydroxy-cis,cis-muconate
RHEA:28932
  • MetaCyc:GALLATE-DEGRADATION-II-PWY

    Source assertion: GALLATE-DIOXYGENASE-RXN is the stated predecessor of RXN-9983; their checked full equations share the intervening pathway compound.

    Source location/ptools-xml/Pathway/reaction-ordering[Reaction/@frameid='RXN-9983']/predecessor-reactions
(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate => 4-carboxy-2-hydroxy-cis,cis-muconate
RHEA:28932		provides_input_for

Native order and direct material continuity.

4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403
  • MetaCyc:GALLATE-DEGRADATION-II-PWY

    Source assertion: RXN-9983 is the stated predecessor of RXN-2463; their checked full equations share the intervening pathway compound.

    Source location/ptools-xml/Pathway/reaction-ordering[Reaction/@frameid='RXN-2463']/predecessor-reactions
4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403		provides_input_for

Native order and direct material continuity.

2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936
  • MetaCyc:GALLATE-DEGRADATION-II-PWY

    Source assertion: RXN-2463 is the stated predecessor of RXN-2464; their checked full equations share the intervening pathway compound.

    Source location/ptools-xml/Pathway/reaction-ordering[Reaction/@frameid='RXN-2464']/predecessor-reactions
protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-2701		catalyzes

Historical purified Comamonas/Pseudomonas testosteroni protocatechuate 4,5-dioxygenase gallate activity (1972/1982 references). The native complex identity is retained; modern sequence cross-references do not establish the historical assayed sequence.

3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928
  • MetaCyc:ENZRXN-11007

    Source assertion: ENZRXN-11007 assigns CPLX-2701 to GALLATE-DIOXYGENASE-RXN. Native evidence: EV-EXP-IDA-PURIFIED-PROTEIN.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
HMG aldolase
MetaCyc:CPLX-7917		catalyzes

Historical Comamonas CHA aldolase activity in crude or partly purified preparations. The source-native complex and its historical evidence remain distinct from current genome proteins.

2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936
  • MetaCyc:ENZRXN-11041

    Source assertion: ENZRXN-11041 assigns CPLX-7917 to RXN-2464. Native evidence: EV-EXP-IDA-UNPURIFIED-PROTEIN,EV-EXP-IDA-PART-PURIFIED-PROTEIN.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) in E. coli BL21(DE3), purified by POROS chromatography; initiating methionine processed. Full terminal construct sequence and any vector-derived residues were not established.

Qualification evidence
  • PMID:16030198

    Source assertion: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host.

    Source locationPMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts
  • PMID:16030198
    cells harboring pETB
    Source locationPMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)
  • PMID:16030198
    appeared to be processed
    Source locationPMC1196043 Results, Purification of DesB, first paragraph, initiating methionine sentence following the N-terminal sequence
catalyzes

Purified SYK-6 DesB produced in E. coli BL21(DE3) with pETB/pET21a(+). The encoded 418-residue reference has a processed initiating methionine in the assayed preparation. Gallate-dependent oxygen consumption gives Vmax 42.7 ± 2.4 U/mg and Km 66.9 ± 9.3 µM (2005 Table 2). Late acidified GC-MS supports OMA enol but does not alone determine the immediate keto product; native formal chemistry and the independent GalA product work supply that distinction. In the separate 500 µM substrate screen (30 °C, pH 8.5), pyrogallol supported only 0.11 U/mg versus gallate 41.8; its product was not established. Tested negative substrates are conditional assay negatives.

3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928
  • MetaCyc:ENZRXN-16936

    Source assertion: ENZRXN-16936 assigns CPLX-7918 to GALLATE-DIOXYGENASE-RXN. Native evidence: EV-EXP-IDA-PURIFIED-PROTEIN-HH.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
gallate dioxygenase
MetaCyc:CPLX-7918		has_cofactor

Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.

iron(2+)
CHEBI:29033
  • MetaCyc:ENZRXN-16936

    Source assertion: ENZRXN-16936 assigns cofactor FE+2 to enzyme CPLX-7918; preserve native citation and assay limits.

    Source location/ptools-xml/Enzymatic-Reaction/cofactor[Compound/@frameid='FE+2']
4-oxalomesaconate tautomerase
MetaCyc:G1G01-2697-MONOMER

experimental construct: 2011 KT2440 GalD expressed from pETGalDc in E. coli BL21(DE3), assayed in partly purified extract. This is separate from the explicitly His6-retaining 2016 GalD preparation.

Qualification evidence
  • PMID:21219457
    pETGalDc
    Source location2011 publisher Figure 3 caption line 249 and Results line 253; Methods lines 349, 365–366 and 376–377 (construct, BL21(DE3) expression, extract preparation). Retained main-text transcription; original spectra and Table S1 were inaccessible.
catalyzes

KT2440 GalD in partly purified E. coli BL21(DE3)/pETGalDc extract converts keto OMA to the E enol. The 2011 NMR text reports 6.73 ppm and no approximately 7.38 ppm Z-enol signal; original spectra were not visually accessible. This enzymatic acceleration coexists with spontaneous tautomerization; residual growth of the galD mutant is concentration dependent.

(1E)-4-oxobut-1-ene-1,2,4-tricarboxylate => 4-carboxy-2-hydroxy-cis,cis-muconate
RHEA:28932
  • MetaCyc:ENZRXN-18372

    Source assertion: ENZRXN-18372 assigns G1G01-2697-MONOMER to RXN-9983. Native evidence: EV-EXP-IDA-PART-PURIFIED-PROTEIN-HH.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
  • PMID:21219457
    (E)-OMAenol
    Source location2011 Figure3 caption publisher line249; original spectrum inaccessible
4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2011 pET29a-derived KT2440 GalB in E. coli BL21(DE3), purified by DEAE and phenyl chromatography. Table 1 reports 244 residues; reference Q88JX8 has 258. Complete insert/termini remain unresolved; the separate 2016 crystal is 240 residues.

Qualification evidence
  • MetaCyc:ENZRXN-18373

    Source assertion: The activity has purified heterologous-host evidence for CPLX-8178 linked to PMID21219457.

    Source location/ptools-xml/Enzymatic-Reaction/evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]
  • PMID:21219457

    Source assertion: Table 1 lists GalB as a 244-residue, 27.4-kDa protein in the gal cluster. This does not identify the complete recombinant insert with the independently deposited 258-residue reference.

    Source location2011 Table1, galB row: protein size and predicted molecular mass columns
  • PMID:21219457
    pETGalB
    Source location2011 Results GalB publisher line274: E. coli BL21(DE3)(pETGalB); Methods GalB expression and purification
catalyzes

Purified 2011 pET29a-derived GalB from E. coli BL21(DE3) hydrates the E enol, not fresh keto OMA. Primary Km 85 µM and Vmax 76 µmol/min/mg apply to enol hydration, despite the native XML attaching them to CHA. Table 1 reports a 244-residue protein, whereas the deposited reference is 258 residues; the complete insert is unresolved. The 2016 240-residue crystallization construct does not settle this discrepancy. The formal CHA identifier leaves absolute stereochemistry unspecified.

4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403
  • MetaCyc:ENZRXN-18373

    Source assertion: ENZRXN-18373 assigns CPLX-8178 to RXN-2463. Native evidence: EV-EXP-IDA-PURIFIED-PROTEIN-HH.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
4-oxalmesaconate hydratase
MetaCyc:CPLX-8178		has_cofactor

Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.

zinc(2+)
CHEBI:29105
  • MetaCyc:ENZRXN-18373

    Source assertion: ENZRXN-18373 assigns cofactor ZN+2 to enzyme CPLX-8178; preserve native citation and assay limits.

    Source location/ptools-xml/Enzymatic-Reaction/cofactor[Compound/@frameid='ZN+2']
4-oxalomesaconate hydratase
MetaCyc:CPLX-2781		catalyzes

Historical purified ProH hydratase from phthalate-grown Pseudomonas straminea/ochraceae; source-native identity and reversible activity are retained. No whole-route occurrence is inferred from this single enzyme.

4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403
  • MetaCyc:ENZRXN-3364

    Source assertion: ENZRXN-3364 assigns CPLX-2781 to RXN-2463. Native evidence: EV-EXP-IDA-PURIFIED-PROTEIN.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
HMG aldolase
MetaCyc:CPLX-2782		catalyzes

Source-native ProA CHA aldolase annotation combines purified native and heterologous-host evidence. These historical preparations are not assumed to equal every current sequence cross-reference.

2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936
  • MetaCyc:ENZRXN-3365

    Source assertion: ENZRXN-3365 assigns CPLX-2782 to RXN-2464. Native evidence: EV-EXP-IDA-PURIFIED-PROTEIN,EV-EXP-IDA-PURIFIED-PROTEIN-HH.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
HMG aldolase
MetaCyc:CPLX-2782		has_cofactor

Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.

magnesium(2+)
CHEBI:18420
  • MetaCyc:ENZRXN-3365

    Source assertion: ENZRXN-3365 assigns cofactor MG+2 to enzyme CPLX-2782; preserve native citation and assay limits.

    Source location/ptools-xml/Enzymatic-Reaction/cofactor[Compound/@frameid='MG+2']
4-carboxy-4-hydroxy-2-oxoadipate aldolase
MetaCyc:CPLX-2865		catalyzes

Source-native SYK-6 LigK CHA aldolase activity. The historical/current sequence and taxonomy identifiers are not silently equated; its additional oxaloacetate decarboxylation is outside the four formal members.

2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936
  • MetaCyc:ENZRXN-3627

    Source assertion: ENZRXN-3627 assigns CPLX-2865 to RXN-2464. Native evidence: EV-EXP-IDA-PURIFIED-PROTEIN.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
4-carboxy-4-hydroxy-2-oxoadipate aldolase
MetaCyc:CPLX-2865		has_cofactor

Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.

magnesium(2+)
CHEBI:18420
  • MetaCyc:ENZRXN-3627

    Source assertion: ENZRXN-3627 assigns cofactor MG+2 to enzyme CPLX-2865; preserve native citation and assay limits.

    Source location/ptools-xml/Enzymatic-Reaction/cofactor[Compound/@frameid='MG+2']
HMG aldolase
MetaCyc:CPLX-2881		catalyzes

Historical purified Pseudomonas putida CHA aldolase, distinct from the sequence-defined KT2440 GalC. The source also discusses HMG specificity; that does not fix CHA absolute stereochemistry.

2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936
  • MetaCyc:ENZRXN-3641

    Source assertion: ENZRXN-3641 assigns CPLX-2881 to RXN-2464. Native evidence: EV-EXP-IDA-PURIFIED-PROTEIN,EV-EXP-IDA-PURIFIED-PROTEIN-NH.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
HMG aldolase
MetaCyc:CPLX-2881		has_cofactor

Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.

manganese(2+)
CHEBI:29035
  • MetaCyc:ENZRXN-3641

    Source assertion: ENZRXN-3641 assigns cofactor MN+2 to enzyme CPLX-2881; preserve native citation and assay limits.

    Source location/ptools-xml/Enzymatic-Reaction/cofactor[Compound/@frameid='MN+2']
HMG aldolase
MetaCyc:CPLX-2881		has_cofactor

Native cofactor annotation on this exact source enzyme. This is not an exclusive physiological metal assignment; preparation-specific reconstitution is recorded separately.

magnesium(2+)
CHEBI:18420
  • MetaCyc:ENZRXN-3641

    Source assertion: ENZRXN-3641 assigns cofactor MG+2 to enzyme CPLX-2881; preserve native citation and assay limits.

    Source location/ptools-xml/Enzymatic-Reaction/cofactor[Compound/@frameid='MG+2']
4-hydroxy-4-methyl-2-oxoglutarate aldolase
MetaCyc:CPLX1G01-155

experimental construct: 2011 KT2440 GalC expressed in E. coli BL21(DE3) from pETGalC, assayed as crude extract. Historical purified GalC/hexamer annotations do not establish the sequence or assembly of this recombinant preparation.

Qualification evidence
  • PMID:21219457
    pETGalC
    Source location2011 publisher Results lines 307–309 and Methods lines 349, 365–366 and 380 (construct, BL21(DE3), crude extract, assay and activities). Retained main-text transcription; original Table S1 and Figure S7 were inaccessible.
catalyzes

KT2440 GalC activity is supported by crude E. coli BL21(DE3)/pETGalC extract and a disrupted KTGAL strain. At 200 µM CHA, 1 mM MgCl2, pH 7, 30 °C the source reports 24.5 µmol/min/mg in recombinant extract and 2.1 in KTGAL, with no detected mutant activity. Whole-cluster complementation and polar effects do not prove isolated-gene sufficiency. Historical purified/homohexamer evidence remains a separate source annotation.

2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936
  • MetaCyc:ENZRXN1G01-1112

    Source assertion: ENZRXN1G01-1112 assigns CPLX1G01-155 to RXN-2464. Native evidence: EV-EXP-IMP-REACTION-BLOCKED,EV-EXP-IDA-UNPURIFIED-PROTEIN-HH,EV-EXP-IDA-PURIFIED-PROTEIN-NH.

    Source location/ptools-xml/Enzymatic-Reaction/enzyme; /ptools-xml/Enzymatic-Reaction/reaction
4-hydroxy-4-methyl-2-oxoglutarate aldolase
MetaCyc:CPLX1G01-155		has_cofactor

Native Mn2+ annotation for the GalC source instance reflects historical purified-enzyme evidence. It does not establish Mn occupancy in the 2011 KT2440 crude recombinant assay, which contained MgCl2.

manganese(2+)
CHEBI:29035
  • MetaCyc:ENZRXN1G01-1112

    Source assertion: ENZRXN1G01-1112 assigns cofactor MN+2 to enzyme CPLX1G01-155; preserve native citation and assay limits.

    Source location/ptools-xml/Enzymatic-Reaction/cofactor[Compound/@frameid='MN+2']
protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-2701		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

protocatechuate 4,5-dioxygenase α subunit
MetaCyc:MONOMER-3165
  • MetaCyc:CPLX-2701

    Source assertion: CPLX-2701 has component MONOMER-3165 with native coefficient 4.

    Source location/ptools-xml/Protein/component[Protein/@frameid='MONOMER-3165']
protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-2701		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

protocatechuate 4,5-dioxygenase β subunit
MetaCyc:MONOMER-3166
  • MetaCyc:CPLX-2701

    Source assertion: CPLX-2701 has component MONOMER-3166 with native coefficient 4.

    Source location/ptools-xml/Protein/component[Protein/@frameid='MONOMER-3166']
4-oxalomesaconate hydratase
MetaCyc:CPLX-2781		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

subunit of 4-oxalomesaconate hydratase
MetaCyc:MONOMER-3243
  • MetaCyc:CPLX-2781

    Source assertion: CPLX-2781 has component MONOMER-3243 with native coefficient 2.

    Source location/ptools-xml/Protein/component[Protein/@frameid='MONOMER-3243']
HMG aldolase
MetaCyc:CPLX-2782		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

4-oxalocitramalate aldolase subunit
MetaCyc:MONOMER-3244
  • MetaCyc:CPLX-2782

    Source assertion: CPLX-2782 has component MONOMER-3244 with native coefficient 6.

    Source location/ptools-xml/Protein/component[Protein/@frameid='MONOMER-3244']
4-carboxy-4-hydroxy-2-oxoadipate aldolase
MetaCyc:CPLX-2865		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

LigK
MetaCyc:MONOMER-3469
  • MetaCyc:CPLX-2865

    Source assertion: CPLX-2865 has component MONOMER-3469 with native coefficient 6.

    Source location/ptools-xml/Protein/component[Protein/@frameid='MONOMER-3469']
HMG aldolase
MetaCyc:CPLX-2881		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

subunit of 4-hydroxy-4-methyl-2-oxoglutarate aldolase
MetaCyc:MONOMER-3481
  • MetaCyc:CPLX-2881

    Source assertion: CPLX-2881 has component MONOMER-3481 with native coefficient 6.

    Source location/ptools-xml/Protein/component[Protein/@frameid='MONOMER-3481']
HMG aldolase
MetaCyc:CPLX-7917		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

HMG aldolase monomer
MetaCyc:MONOMER-3184
  • MetaCyc:CPLX-7917

    Source assertion: CPLX-7917 has component MONOMER-3184 with native coefficient 6.

    Source location/ptools-xml/Protein/component[Protein/@frameid='MONOMER-3184']
gallate dioxygenase
MetaCyc:CPLX-7918		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

gallate dioxygenase monomer
MetaCyc:MONOMER-15119
  • MetaCyc:CPLX-7918

    Source assertion: CPLX-7918 has component MONOMER-15119 with native coefficient 2.

    Source location/ptools-xml/Protein/component[Protein/@frameid='MONOMER-15119']
4-oxalmesaconate hydratase
MetaCyc:CPLX-8178		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

4-oxalmesaconate hydratase monomer
MetaCyc:G1G01-2699-MONOMER
  • MetaCyc:CPLX-8178

    Source assertion: CPLX-8178 has component G1G01-2699-MONOMER with native coefficient 6.

    Source location/ptools-xml/Protein/component[Protein/@frameid='G1G01-2699-MONOMER']
4-hydroxy-4-methyl-2-oxoglutarate aldolase
MetaCyc:CPLX1G01-155		has_part

Component is not asserted independently catalytic. Historical source subunit is not automatically equal to current cross-referenced sequence.

4-hydroxy-4-methyl-2-oxoglutarate aldolase aldolase
MetaCyc:G1G01-2698-MONOMER
  • MetaCyc:CPLX1G01-155

    Source assertion: CPLX1G01-155 has component G1G01-2698-MONOMER with native coefficient 6.

    Source location/ptools-xml/Protein/component[Protein/@frameid='G1G01-2698-MONOMER']
oxaloacetate(2-)
CHEBI:16452		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

4-carboxy-4-hydroxy-2-oxoadipate aldolase
MetaCyc:CPLX-2865
  • MetaCyc:REG-10400

    Source assertion: REG-10400 has regulator OXALACETIC_ACID, mode -, target activity ENZRXN-3627 (enzyme CPLX-2865). Mechanism: COMPETITIVE.

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
N-ethylmaleimide
MetaCyc:N-ETHYLMALEIMIDE		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

4-carboxy-4-hydroxy-2-oxoadipate aldolase
MetaCyc:CPLX-2865
  • MetaCyc:REG-10401

    Source assertion: REG-10401 has regulator N-ETHYLMALEIMIDE, mode -, target activity ENZRXN-3627 (enzyme CPLX-2865).

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
oxaloacetate(2-)
CHEBI:16452		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

HMG aldolase
MetaCyc:CPLX-2881
  • MetaCyc:REG-10724

    Source assertion: REG-10724 has regulator OXALACETIC_ACID, mode -, target activity ENZRXN-3641 (enzyme CPLX-2881). Mechanism: NONCOMPETITIVE.

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
mercury(2+)
CHEBI:16793		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

4-carboxy-4-hydroxy-2-oxoadipate aldolase
MetaCyc:CPLX-2865
  • MetaCyc:REG-12626

    Source assertion: REG-12626 has regulator HG+2, mode -, target activity ENZRXN-3627 (enzyme CPLX-2865).

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
mercury(2+)
CHEBI:16793		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

4-oxalomesaconate hydratase
MetaCyc:CPLX-2781
  • MetaCyc:REG-12629

    Source assertion: REG-12629 has regulator HG+2, mode -, target activity ENZRXN-3364 (enzyme CPLX-2781).

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
mercury(2+)
CHEBI:16793		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

HMG aldolase
MetaCyc:CPLX-2782
  • MetaCyc:REG-12638

    Source assertion: REG-12638 has regulator HG+2, mode -, target activity ENZRXN-3365 (enzyme CPLX-2782).

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
zinc(2+)
CHEBI:29105		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

4-oxalomesaconate hydratase
MetaCyc:CPLX-2781
  • MetaCyc:REG-12669

    Source assertion: REG-12669 has regulator ZN+2, mode -, target activity ENZRXN-3364 (enzyme CPLX-2781).

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
oxaloacetate(2-)
CHEBI:16452		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

HMG aldolase
MetaCyc:CPLX-2782
  • MetaCyc:REG-8391

    Source assertion: REG-8391 has regulator OXALACETIC_ACID, mode -, target activity ENZRXN-3365 (enzyme CPLX-2782). Mechanism: COMPETITIVE.

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
p-chloromercuribenzoate
MetaCyc:CPD-29		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

HMG aldolase
MetaCyc:CPLX-2782
  • MetaCyc:REG-8393

    Source assertion: REG-8393 has regulator CPD-29, mode -, target activity ENZRXN-3365 (enzyme CPLX-2782).

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
p-chloromercuribenzoate
MetaCyc:CPD-29		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

4-oxalomesaconate hydratase
MetaCyc:CPLX-2781
  • MetaCyc:REG-9531

    Source assertion: REG-9531 has regulator CPD-29, mode -, target activity ENZRXN-3364 (enzyme CPLX-2781).

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
copper sulfate
MetaCyc:CUSO4		inhibits

Source assay effect on this catalyst only, not global inhibition of the pathway.

4-oxalomesaconate hydratase
MetaCyc:CPLX-2781
  • MetaCyc:REG-9532

    Source assertion: REG-9532 has regulator CUSO4, mode -, target activity ENZRXN-3364 (enzyme CPLX-2781).

    Source location/ptools-xml/Regulation/regulator; mode; regulated-entity
2-hydroxy-4-oxobutane-1,2,4-tricarboxylate => oxaloacetate + pyruvate
RHEA:28936		provides_input_for

The final aldolase reaction produces oxaloacetate, the metabolite explicitly linked by this native pathway to the prokaryotic TCA pathway. This is a material interface, not an additional formal gallate reaction.

TCA cycle I (prokaryotic)
MetaCyc:TCA
  • MetaCyc:GALLATE-DEGRADATION-II-PWY

    Source assertion: The native pathway-link identifies oxaloacetate as the interface compound linking this pathway to TCA.

    Source location/ptools-xml/Pathway/pathway-link[Compound/@frameid="OXALACETIC_ACID"]/link-target
MetaCyc:GALLATE-DEGRADATION-II-PWYpart_of

Syringate parent context; no upstream route reactions added to the four formal members.

syringate degradation
MetaCyc:PWY-6339
  • MetaCyc:GALLATE-DEGRADATION-II-PWY

    Source assertion: The native object lists PWY-6339 as its super-pathway and in-pathway parent.

    Source location/ptools-xml/Pathway/super-pathways; in-pathway
gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established. The encoded chain is 420 aa; Edman results suggest 419 aa after initiator-Met processing.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • NCBIProtein:AAN68130.2

    Source assertion: The PP_2518 CDS names protein AAN68130.2 and supplies a 420-residue translation beginning MARIIGG.

    Source locationAE015451.2 CDS complement(2865514..2866776), /locus_tag, /protein_id and /translation.
  • PMID:16030014
    appeared to be processed.
    Source locationResults cloning/expression, p.35385; gal2005_methods-successor-web.json L1011-L1019, exact phrase at L1015.
catalyzes

GalA catalyzes gallate ring cleavage in the purified recombinant assay. The 2005 late product analysis alone does not distinguish the immediate keto/enol product; formal equation selection depends on the native/Rhea and 2011 chemistry reconciliation.

3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928
  • MetaCyc:ENZRXN-18371

    Source assertion: ENZRXN-18371 connects enzyme CPLX-8177 to GALLATE-DIOXYGENASE-RXN and records a purified heterologous-host assay with PMID16030014.

    Source location/ptools-xml/Enzymatic-Reaction[@frameid="ENZRXN-18371"]/{enzyme,reaction,evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]}
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • PMID:21219457
    OMAketo
    Source location2011 fresh GalA product NMR Results line243 and Figure3 caption lines248–250; original figure inaccessible
gallate dioxygenase
MetaCyc:CPLX-8177		has_cofactor

Native ENZRXN-18371 records Fe2+ as GalA cofactor with the 2005 publication. This native annotation does not fix iron occupancy or imply that other ions fully restore activity. The recombinant pretreatment/rescue observations are separate qualified edges.

iron(2+)
CHEBI:29033
  • MetaCyc:ENZRXN-18371

    Source assertion: The cofactor slot of ENZRXN-18371 identifies FE+2 and cites PUB-16030014.

    Source location/ptools-xml/Enzymatic-Reaction[@frameid="ENZRXN-18371"]/cofactor
gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
has_part

The native component relation and the recombinant assembly experiment are joined here with the preparation qualification. The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. Analytical ultracentrifugation found a predominant species representing about 75% of loading protein, with corrected sedimentation coefficient 7.29 ± 0.10 S and equilibrium mass 132 ± 12 kDa. Comparison with calculated monomer mass 47.644 kDa supports a dominant homotrimer. The preparation was not shown to be exclusively trimeric, and the native component slot supplies no stoichiometric coefficient.

gallate dioxygenase monomer
MetaCyc:G1G01-2702-MONOMER
  • MetaCyc:CPLX-8177

    Source assertion: The component slot of CPLX-8177 points to G1G01-2702-MONOMER; this slot has no numeric coefficient.

    Source location/ptools-xml/Protein[@frameid="CPLX-8177"]/component/Protein[@frameid="G1G01-2702-MONOMER"]
  • PMID:16030014
    globular trimer
    Source locationResults, p.35387, sedimentation paragraph; gal2005_rg_rest2-web.json L1446-L1461, phrase L1459.
ethylenediaminetetraacetic acid
CHEBI:4735		inhibits

The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. 1 mM EDTA left less than 1% of untreated activity. This is an in-vitro exposure result; no cellular flux or specific metal-occupancy mechanism is asserted. The source names EDTA without a salt or protonation specification. CHEBI:4735 is the canonical named parent-acid reference for that reagent; this normalization does not identify the assayed microspecies, supplied salt, charge or stoichiometry.

gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • PMID:16030014
    ⬍1% of untreated enzyme
    Source locationResults, p.35386; gal2005_rg_rest1-web.json L1324-L1329. Protocol: gal2005_methods-successor-web.json L751-L814.
  • CHEBI:4735

    Source assertion: CHEBI:4735 names ethylenediaminetetraacetic acid and lists EDTA and EDTA (chelating agent) as synonyms; its chemical data describe the neutral parent acid.

    Source locationCHEBI-4735.json $.{chebi_accession,ascii_name,names.SYNONYM[ascii_name="EDTA"],names.SYNONYM[ascii_name="EDTA (chelating agent)"],chemical_data,default_structure}
2,2'-bipyridine
CHEBI:30351		inhibits

The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. 2 mM 2,2′-dipyridyl abolished detectable activity. This is an in-vitro exposure result; no cellular flux or specific metal-occupancy mechanism is asserted.

gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • PMID:16030014
    abolished
    Source locationResults, p.35386; gal2005_rg_rest1-web.json L1333-L1339. Protocol: gal2005_methods-successor-web.json L751-L814.
hydrogen peroxide
CHEBI:16240		inhibits

The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. 1 mM hydrogen peroxide left 15% of untreated activity. This is an in-vitro exposure result; no cellular flux or specific metal-occupancy mechanism is asserted.

gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • PMID:16030014
    activity to 15%
    Source locationResults, p.35386; gal2005_rg_rest2-web.json L1396-L1400. Protocol: gal2005_methods-successor-web.json L751-L814.
iron(2+)
CHEBI:29033		activates

The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. After EDTA or dipyridyl pretreatment, 2 mM Fe2+ supplied as FeSO4 restored activity fully following a further 30 min at 4 °C. The two chelator preparations are not reported separately in these outcomes; this conditional rescue is not activation of an untreated enzyme. Metal salt delivery is retained without equating a salt with the ion endpoint.

gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • PMID:16030014
    recovered
    Source locationResults, p.35386; gal2005_rg_rest1-web.json L1341-L1379. Protocol: gal2005_methods-successor-web.json L751-L814.
manganese(2+)
CHEBI:29035		activates

The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. After chelator pretreatment, 2 mM Mn2+ supplied as MnSO4 restored 8% of untreated activity following a further 30 min at 4 °C. The two chelator preparations are not reported separately in these outcomes; this conditional rescue is not activation of an untreated enzyme. Metal salt delivery is retained without equating a salt with the ion endpoint.

gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • PMID:16030014
    recovered 8,
    Source locationResults, p.35386; gal2005_rg_rest1-web.json L1383-L1396: first result in ordered Mn2+, Cu2+, Co2+ list. Protocol: gal2005_methods-successor-web.json L751-L814.
copper(2+)
CHEBI:29036		activates

The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. After chelator pretreatment, 2 mM Cu2+ supplied as CuCl2 restored 9% of untreated activity following a further 30 min at 4 °C. The two chelator preparations are not reported separately in these outcomes; this conditional rescue is not activation of an untreated enzyme. Metal salt delivery is retained without equating a salt with the ion endpoint.

gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • PMID:16030014
    9,
    Source locationResults, p.35386; gal2005_rg_rest1-web.json L1383-L1396: second result in ordered Mn2+, Cu2+, Co2+ list. Protocol: gal2005_methods-successor-web.json L751-L814.
cobalt(2+)
CHEBI:48828		activates

The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. After chelator pretreatment, 2 mM Co2+ supplied as CoCl2 restored 5% of untreated activity following a further 30 min at 4 °C. The two chelator preparations are not reported separately in these outcomes; this conditional rescue is not activation of an untreated enzyme. Metal salt delivery is retained without equating a salt with the ion endpoint.

gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • PMID:16030014
    5% activity
    Source locationResults, p.35386; gal2005_rg_rest1-web.json L1383-L1396: third result in ordered Mn2+, Cu2+, Co2+ list. Protocol: gal2005_methods-successor-web.json L751-L814.
sodium ascorbate
CHEBI:113451		activates

The source reports apparent initiator-Met removal from the 420-aa encoded chain, consistent with a 419-aa processed enzyme; other recombinant terminal changes are unverified. The purified enzyme was 1 mg/ml in 100 mM Tris-HCl pH 7.0, treated for 30 min at 4 °C before activity was initiated with 250 micromolar gallate. Following the 1 mM peroxide pretreatment, 10 mM sodium ascorbate and a further 30 min at 4 °C restored activity to 50% of untreated enzyme. This is partial rescue of the peroxide-exposed preparation. It does not establish constitutive activation or an in-vivo effect.

gallate dioxygenase
MetaCyc:CPLX-8177

experimental construct: Purified KT2440 GalA produced with pETGalA in E. coli BL21(DE3), following ultracentrifugation and phenyl-Sepharose purification. The complete recombinant insert/terminal sequence was not established.

Qualification evidence
  • PMID:16030014

    Source assertion: Table 1 identifies gallate dioxygenase purified from E. coli carrying pETGalA; its phenyl-Sepharose row reports specific activity 42.7 micromol/min/mg.

    Source locationTable 1, p.35385, title plus purification step 3/specific-activity column; gal2005_rg_rest1-web.json L1212-L1233. Host OCR caveat: title BLB21(DE3), Figure 1 caption L1206 BL21(DE3).
  • PMID:16030014
    partially restored (50%
    Source locationResults, p.35386; gal2005_rg_rest2-web.json L1396-L1406; sodium salt specified in methods L804-L814. Protocol: gal2005_methods-successor-web.json L751-L814.
HTH-type transcriptional regulator GalR
UniProtKB:Q88JX7

experimental construct: GalR supplied in an additional plasmid copy in P. putida KT2440, with PP_RS13150-RFP on pIK002_T; the promoter-only pIK002A_T control retains endogenous GalR. Readout: 6 h after 1.25 mM gallate in LB/MM. Native target-gene transcription and direct GalR binding were not measured.

Qualification evidence
  • PMID:36724292
    extra copy of galR gene
    Source locationPMC9942251 XML body paragraph ordinal 10 (zero-based 9); experimental comparison in body paragraph ordinal 8 (zero-based 7) and Figure 2B.
regulates

Positive transcriptional regulation of the galb promoter/gene context is inferred from the KT2440 RFP reporter comparison. The promoter PP_RS13150 was assayed on pIK002_T versus pIK002A_T: 45/248-fold induction with the extra-copy construct versus 1.7/6.3-fold with the promoter-only vector in LB/MM, measured 6 h after 1.25 mM gallic acid in the source experiment. The promoter-only host retains its endogenous galR; the comparison is an additional plasmid copy, not a galR-null rescue. This does not directly measure native target-gene RNA, exact promoter occupancy, GalR ligand binding, or GalB/GalT protein activation. The reference GalR sequence is independently grounded; the exact 2023 plasmid insert was not sequenced here.

PP_2515
KEGG:ppu:PP_2515
  • PMID:36724292
    transcriptional activator for both promoters
    Source locationPMC9942251 XML body paragraph ordinal 10 (zero-based 9), GalR/promoter results; Figure 2B and Supplementary Figure S2 establish reporter context.
HTH-type transcriptional regulator GalR
UniProtKB:Q88JX7

experimental construct: GalR supplied in an additional plasmid copy in P. putida KT2440, with PP_RS13170-RFP on pIK014_T; the promoter-only pIK014A_T control retains endogenous GalR. Readout: 6 h after 1.25 mM gallate in LB/MM. Native target-gene transcription and direct GalR binding were not measured.

Qualification evidence
  • PMID:36724292
    extra copy of galR gene
    Source locationPMC9942251 XML body paragraph ordinal 10 (zero-based 9); experimental comparison in body paragraph ordinal 8 (zero-based 7) and Figure 2B.
regulates

Positive transcriptional regulation of the galt promoter/gene context is inferred from the KT2440 RFP reporter comparison. The promoter PP_RS13170 was assayed on pIK014_T versus pIK014A_T: 9.7/72.3-fold induction with the extra-copy construct versus 2.5/45.6-fold with the promoter-only vector in LB/MM, measured 6 h after 1.25 mM gallic acid in the source experiment. The promoter-only host retains its endogenous galR; the comparison is an additional plasmid copy, not a galR-null rescue. This does not directly measure native target-gene RNA, exact promoter occupancy, GalR ligand binding, or GalB/GalT protein activation. The reference GalR sequence is independently grounded; the exact 2023 plasmid insert was not sequenced here.

PP_2519
KEGG:ppu:PP_2519
  • PMID:36724292
    transcriptional activator for both promoters
    Source locationPMC9942251 XML body paragraph ordinal 10 (zero-based 9), GalR/promoter results; Figure 2B and Supplementary Figure S2 establish reporter context.
protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916		has_part

Native SYK-6 complex component; no coefficient is present in the MetaCyc component slot. This does not assert that unmodified native material was used in the 2013 or 2015 assays.

protocatechuate 4,5-dioxygenase alpha chain
MetaCyc:MONOMER-15116
  • MetaCyc:CPLX-7916

    Source assertion: CPLX-7916 has a component slot referring to MONOMER-15116; that slot supplies no coefficient.

    Source location/ptools-xml/Protein[@frameid="CPLX-7916"]/component/Protein[@frameid="MONOMER-15116"]
protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916		has_part

Native SYK-6 complex component; no coefficient is present in the MetaCyc component slot. This does not assert that unmodified native material was used in the 2013 or 2015 assays.

protocatechuate 4,5-dioxygenase beta chain
MetaCyc:MONOMER-15117
  • MetaCyc:CPLX-7916

    Source assertion: CPLX-7916 has a component slot referring to MONOMER-15117; that slot supplies no coefficient.

    Source location/ptools-xml/Protein[@frameid="CPLX-7916"]/component/Protein[@frameid="MONOMER-15117"]
protocatechuate 4,5-dioxygenase beta chain
MetaCyc:MONOMER-15117		has_cofactor

The issuing UniProt LigB reference annotation identifies Fe2+ as a cofactor. It does not fix occupancy, prove gallate-specific metal rescue, or establish the exact synthetic construct identity.

iron(2+)
CHEBI:29033
  • UniProtKB:P22636

    Source assertion: The COFACTOR comment lists Fe(2+) with ChEBI identifier CHEBI:29033.

    Source location$.comments[?(@.commentType=="COFACTOR")].cofactors[0].cofactorCrossReference
2,3,4-trihydroxybenzoic acid
CHEBI:88714		inhibits

Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 2,3,4-trihydroxybenzoic acid reduced PCA-dependent O2 consumption by 95% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.

protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916

experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.

Qualification evidence
  • PMID:23977959
    His-tag cleaved
    Source location#S4/#P16; retained original HTML line1422, before Source-Q purification
  • PMID:23977959

    Source assertion: Table 2 records 95% inhibition for 2,3,4-trihydroxybenzoic acid. Footnote b specifies 500 µM PCA and 1 mM inhibitor in air-saturated 50 mM Tris, pH 7.5, 25 °C.

    Source location#T2 row "2,3,4-trihydroxybenzoic acid", column "% inhibition", footnote b
2,3-dihydroxybenzoic acid
CHEBI:18026		inhibits

Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 2,3-dihydroxybenzoic acid reduced PCA-dependent O2 consumption by 36% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.

protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916

experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.

Qualification evidence
  • PMID:23977959
    His-tag cleaved
    Source location#S4/#P16; retained original HTML line1422, before Source-Q purification
  • PMID:23977959

    Source assertion: Table 2 records 36% inhibition for 2,3-dihydroxybenzoic acid. Footnote b specifies 500 µM PCA and 1 mM inhibitor in air-saturated 50 mM Tris, pH 7.5, 25 °C.

    Source location#T2 row "2,3-dihydroxybenzoic acid", column "% inhibition", footnote b
4-nitrocatechol
CHEBI:16318		inhibits

Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 4-nitrocatechol reduced PCA-dependent O2 consumption by 31% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.

protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916

experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.

Qualification evidence
  • PMID:23977959
    His-tag cleaved
    Source location#S4/#P16; retained original HTML line1422, before Source-Q purification
  • PMID:23977959

    Source assertion: Table 2 records 31% inhibition for 4-nitrocatechol. Footnote b specifies 500 µM PCA and 1 mM inhibitor in air-saturated 50 mM Tris, pH 7.5, 25 °C.

    Source location#T2 row "4-nitrocatechol", column "% inhibition", footnote b
4-methylcatechol
CHEBI:17254		inhibits

Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 4-methylcatechol reduced PCA-dependent O2 consumption by 28% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.

protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916

experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.

Qualification evidence
  • PMID:23977959
    His-tag cleaved
    Source location#S4/#P16; retained original HTML line1422, before Source-Q purification
  • PMID:23977959

    Source assertion: Table 2 records 28% inhibition for 4-methylcatechol. Footnote b specifies 500 µM PCA and 1 mM inhibitor in air-saturated 50 mM Tris, pH 7.5, 25 °C.

    Source location#T2 row "4-methylcatechol", column "% inhibition", footnote b
3-methoxycatechol
CHEBI:141700		inhibits

Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). 3-methoxycatechol reduced PCA-dependent O2 consumption by 26% with 500 µM PCA, 1 mM inhibitor and 3–7 nM LigAB in air-saturated 50 mM Tris, pH 7.5, 25 °C (Table 2b/P27). This does not establish a competitive Ki or inhibition of gallate flux. The chemical endpoint follows the neutral parent drawn in SI Figure S2; supplied salt and unique assay microspecies are not assigned.

protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916

experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.

Qualification evidence
  • PMID:23977959
    His-tag cleaved
    Source location#S4/#P16; retained original HTML line1422, before Source-Q purification
  • PMID:23977959

    Source assertion: Table 2 records 26% inhibition for 3-methoxycatechol. Footnote b specifies 500 µM PCA and 1 mM inhibitor in air-saturated 50 mM Tris, pH 7.5, 25 °C.

    Source location#T2 row "3-methoxycatechol", column "% inhibition", footnote b
ethylenediaminetetraacetic acid
CHEBI:4735		inhibits

Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). PCA activity retained 65% after 1 h of EDTA at pH 7.5; the dose is not explicit for that time point. After 24 h at 1 or 2 mM EDTA, 35% remained. A separate 1 mM/2 h treatment retained 50%. Readout: 250 µM PCA (P23/P34). Canonical named-parent normalization does not establish the supplied EDTA salt, assayed neutral acid, unique pH microspecies, charge or stoichiometry.

protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916

experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.

Qualification evidence
  • PMID:23977959
    His-tag cleaved
    Source location#S4/#P16; retained original HTML line1422, before Source-Q purification
  • PMID:23977959
    maintained 65% activity
    Source location#S14/#P34; numerical result in the named treatment sentence; protocol #S7/#P23
2,2'-bipyridine
CHEBI:30351		inhibits

Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). PCA activity retained 50% after 1 h and none after 24 h with 2 mM bipyridine at 4 °C under N2. Readout: 250 µM PCA in 50 mM Tris, pH 7.5 (P23/P34). No gallate assay or reversible physiological effect was demonstrated.

protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916

experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.

Qualification evidence
  • PMID:23977959
    His-tag cleaved
    Source location#S4/#P16; retained original HTML line1422, before Source-Q purification
  • PMID:23977959
    50% activity
    Source location#S14/#P34; numerical result in the named treatment sentence; protocol #S7/#P23
copper(2+)
CHEBI:29036		inhibits

Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). After 1 mM EDTA for 2 h left 50% activity, 2 mM Cu2+ for 1 h lowered residual PCA activity to 8% of untreated. The metal protocol was anaerobic at 4 °C, with a 250 µM PCA readout (P23/P34). Co2+ and Mn2+ did not cause the additional loss. This is not an untreated-enzyme copper effect or a measured gallate-flux effect.

protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916

experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.

Qualification evidence
  • PMID:23977959
    His-tag cleaved
    Source location#S4/#P16; retained original HTML line1422, before Source-Q purification
  • PMID:23977959
    8%
    Source location#S14/#P34; numerical result in the named treatment sentence; protocol #S7/#P23
iron(2+)
CHEBI:29033		activates

Synthetic NCBI-derived SYK-6 ligAB was expressed from pET15b in E. coli BL21; alpha was initially N-His-tagged and beta was untagged. Anaerobic purification included thrombin cleavage (P8–P16). After 2 mM bipyridine stripping for 24 h, 2 mM FeSO4 for 1 h anaerobically at 4 °C restored only 5% of untreated PCA activity. Longer incubation did not improve rescue; other tested metals did not rescue (P23/P34). This is partial rescue of treated material, not general activation of native LigAB or gallate flux.

protocatechuate 4,5-dioxygenase
MetaCyc:CPLX-7916

experimental construct: His-tag-cleaved LigAB preparation. Full synthesized insert and residual thrombin scar were not established.

Qualification evidence
  • PMID:23977959
    His-tag cleaved
    Source location#S4/#P16; retained original HTML line1422, before Source-Q purification
  • PMID:23977959
    5%
    Source location#S14/#P34; numerical result in the named treatment sentence; protocol #S7/#P23
3,4-dihydroxybenzoate
CHEBI:36241		inhibits

Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.

Qualification evidence
  • DOI:10.1016/j.procbio.2021.01.019

    Source assertion: Table 1 row PCA reports 49 ± 4.5% inhibition at 5 mM inhibitor. The experiment measures gallate-dependent oxygen consumption in the recombinant preparation with 1 mM gallate.

    Source locationAuthor-deposited main paper Table1, row PCA, Observed Percent Inhibition (5mM) column; Methods inhibition and Figure3 legend; transcription lines2283-2307.
4-nitrocatechol
CHEBI:16318		inhibits

Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.

Qualification evidence
  • DOI:10.1016/j.procbio.2021.01.019

    Source assertion: Table 1 row 4-Nitrocatechol reports 31.6 ± 8.3% inhibition at 5 mM inhibitor. The experiment measures gallate-dependent oxygen consumption in the recombinant preparation with 1 mM gallate.

    Source locationAuthor-deposited main paper Table1, row 4-Nitrocatechol, Observed Percent Inhibition (5mM) column; Methods inhibition and Figure3 legend; transcription lines2283-2307.
vanillate
CHEBI:16632		inhibits

Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.

Qualification evidence
  • DOI:10.1016/j.procbio.2021.01.019

    Source assertion: Table 1 row Vanilliate reports 24.2 ± 1.6% inhibition at 5 mM inhibitor. The experiment measures gallate-dependent oxygen consumption in the recombinant preparation with 1 mM gallate.

    Source locationAuthor-deposited main paper Table1, row Vanilliate, Observed Percent Inhibition (5mM) column; Methods inhibition and Figure3 legend; transcription lines2283-2307.
2-Methoxy-4-methylphenol
CHEBI:89886		inhibits

Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.

Qualification evidence
  • DOI:10.1016/j.procbio.2021.01.019

    Source assertion: Table 1 row 2-Methoxy-4-methylphenol reports 20.8 ± 6.9% inhibition at 5 mM inhibitor. The experiment measures gallate-dependent oxygen consumption in the recombinant preparation with 1 mM gallate.

    Source locationAuthor-deposited main paper Table1, row 2-Methoxy-4-methylphenol, Observed Percent Inhibition (5mM) column; Methods inhibition and Figure3 legend; transcription lines2283-2307.
syringate
CHEBI:132111		inhibits

Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.

Qualification evidence
  • DOI:10.1016/j.procbio.2021.01.019

    Source assertion: Table 1 row Syringic Acid reports 16.3 ± 2.2% inhibition at 5 mM inhibitor. The experiment measures gallate-dependent oxygen consumption in the recombinant preparation with 1 mM gallate.

    Source locationAuthor-deposited main paper Table1, row Syringic Acid, Observed Percent Inhibition (5mM) column; Methods inhibition and Figure3 legend; transcription lines2283-2307.
vanillin
CHEBI:18346		inhibits

Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.

Qualification evidence
  • DOI:10.1016/j.procbio.2021.01.019

    Source assertion: Table 1 row Vanillin reports 11.3 ± 5.9% inhibition at 5 mM inhibitor. The experiment measures gallate-dependent oxygen consumption in the recombinant preparation with 1 mM gallate.

    Source locationAuthor-deposited main paper Table1, row Vanillin, Observed Percent Inhibition (5mM) column; Methods inhibition and Figure3 legend; transcription lines2283-2307.
syringaldehyde
CHEBI:67380		inhibits

Gallate-dependent oxygen-consumption inhibition in the 2021 DesB-MBP preparation at 5 mM test compound and 1 mM gallate. Source chemical names are normalized using the independently inspected supplement drawings; no physiological flux effect or unique assay microspecies is inferred.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.

Qualification evidence
  • DOI:10.1016/j.procbio.2021.01.019

    Source assertion: Table 1 row Syringealdehyde reports 8.66 ± 0.22% inhibition at 5 mM inhibitor. The experiment measures gallate-dependent oxygen consumption in the recombinant preparation with 1 mM gallate.

    Source locationAuthor-deposited main paper Table1, row Syringealdehyde, Observed Percent Inhibition (5mM) column; Methods inhibition and Figure3 legend; transcription lines2283-2307.
gallate
CHEBI:16918		inhibits

In vitro inhibition at high gallate concentrations in the measured substrate range; gallate remains the substrate at lower concentrations. Not a cellular flux claim.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Anaerobically purified 91.22-kDa DesB-MBP fusion expressed from synthetic desB/pMAL-c5x in E.coli BL21; minor unfused DesB and MBP remain. The complete fusion sequence and exact originating NCBI accession are not supplied.

Qualification evidence
  • DOI:10.1016/j.procbio.2021.01.019
    substrate inhibition
    Source locationResults kinetic characterization, author-deposited maintextline996; explicitly at high concentrations; SI FigureS2 originaldata
ethylenediaminetetraacetic acid
CHEBI:4735		inhibits

500 µM EDTA for 20 h at 0 °C eliminated detected gallate-dependent oxygen consumption. The oxygen-consumption assay used gallate; this is a treatment-specific in vitro effect, not a physiological metal preference. EDTA is normalized to its named parent without assigning the supplied salt or assay microspecies.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.

Qualification evidence
  • PMID:16030198

    Source assertion: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host.

    Source locationPMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts
  • PMID:16030198
    cells harboring pETB
    Source locationPMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)
  • PMID:16030198
    No oxygen consumption
    Source locationPMC1196043 Results, paragraph beginning “In order to investigate the metal ion dependence of DesB”
iron(2+)
CHEBI:29033		activates

After EDTA treatment, 1 mM Fe2+ for 5 min on ice restored 119% of untreated activity. The oxygen-consumption assay used gallate; this is a treatment-specific in vitro effect, not a physiological metal preference. EDTA is normalized to its named parent without assigning the supplied salt or assay microspecies.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.

Qualification evidence
  • PMID:16030198

    Source assertion: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host.

    Source locationPMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts
  • PMID:16030198
    cells harboring pETB
    Source locationPMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)
  • PMID:16030198
    (119%)
    Source locationPMC1196043 Results, paragraph beginning “In order to investigate the metal ion dependence of DesB”
manganese(2+)
CHEBI:29035		activates

After EDTA treatment, 1 mM Mn2+ for 5 min on ice restored 14% of untreated activity. The oxygen-consumption assay used gallate; this is a treatment-specific in vitro effect, not a physiological metal preference. EDTA is normalized to its named parent without assigning the supplied salt or assay microspecies.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.

Qualification evidence
  • PMID:16030198

    Source assertion: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host.

    Source locationPMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts
  • PMID:16030198
    cells harboring pETB
    Source locationPMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)
  • PMID:16030198
    (14%)
    Source locationPMC1196043 Results, paragraph beginning “In order to investigate the metal ion dependence of DesB”
copper(2+)
CHEBI:29036		activates

After EDTA treatment, 1 mM Cu2+ for 5 min on ice restored 7% of untreated activity. The oxygen-consumption assay used gallate; this is a treatment-specific in vitro effect, not a physiological metal preference. EDTA is normalized to its named parent without assigning the supplied salt or assay microspecies.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: 2005 SYK-6 DesB from pETB/pET21a(+) expressed in E. coli BL21(DE3), purified using POROS columns. Initiating methionine was processed; no complete terminal construct sequence is supplied. For metal rescue, the preparation first received 500 µM EDTA for 20 h at 0 °C.

Qualification evidence
  • PMID:16030198

    Source assertion: Table 1 lists pETB as pET21a(+) with a 2.8-kb NdeI-XbaI fragment of pEVK3G carrying desB. A separate E. coli BL21(DE3) row gives its genotype and T7 RNA polymerase/lacUV5 description; the table does not assign pETB to that host.

    Source locationPMC1196043 Table 1, pETB and E. coli BL21(DE3) rows; host assignment is in Methods, Expression of desB in E. coli and preparation of cell extracts
  • PMID:16030198
    cells harboring pETB
    Source locationPMC1196043 Methods, Expression of desB in E. coli and preparation of cell extracts, first paragraph (BL21(DE3)/pETB); Purification of DesB, first paragraph (POROS PI and quaternized PI); Results, Purification of DesB, first paragraph (N-terminus)
  • PMID:16030198
    (7%)
    Source locationPMC1196043 Results, paragraph beginning “In order to investigate the metal ion dependence of DesB”
4-nitrocatechol
CHEBI:16318		inhibits

2018 4-nitrocatechol inhibition of gallate-dependent oxygen consumption: discussion reports 36.6% at fivefold inhibitor excess and Ki 2.3 ± 0.3 mM. The 1 mM gallate assay tested 0.05–20 mM inhibitor. Figure 7 ratio labels conflict with decreasing activity; no binding-site mechanism is established.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Synthetic desB in pMAL-c5x, expressed in E. coli BL21(DE3); anaerobically purified 91.22-kDa MBP fusion with minor cleaved DesB/MBP. Complete originating accession, inserted sequence and fusion junction were not supplied.

Qualification evidence
  • PMID:30346405
    fusion construct
    Source locationPMC6235412 Representative Results paragraph beginning “Protein purification”; Figure 3 fusion material
  • PMID:30346405
    36.6%
    Source locationPMC6235412 Discussion and Figure 6/7; quantitative and label conflicts retained in the author ledger
gallate
CHEBI:16918		inhibits

2018 high-gallate inhibition is supported by Figure 6/Haldane fitting. Figure axis units and discussion-versus-caption kinetic values conflict; retain the qualitative observation in the tested concentration series without averaging the inconsistent values.

gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: Synthetic desB in pMAL-c5x, expressed in E. coli BL21(DE3); anaerobically purified 91.22-kDa MBP fusion with minor cleaved DesB/MBP. Complete originating accession, inserted sequence and fusion junction were not supplied.

Qualification evidence
  • PMID:30346405
    fusion construct
    Source locationPMC6235412 Representative Results paragraph beginning “Protein purification”; Figure 3 fusion material
  • PMID:30346405
    substrate inhibition
    Source locationPMC6235412 Discussion and Figure 6/7; quantitative and label conflicts retained in the author ledger
iron(2+)
CHEBI:29033		activates

In the 2016 metal-substitution assay, Fe2+ supported 100 ± 4.6% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Fe2+: 100 ± 4.6% relative activity, Apo: 4.0 ± 0.33%, and Fe2+: 100 ± 4.6%. Its footnote defines the untreated native preparation separately.

    Source locationPMC4817193 #T2, Fe2+, Apo and Fe2+ rows; relative-activity column and footnote
cobalt(2+)
CHEBI:48828		activates

In the 2016 metal-substitution assay, Co2+ supported 79 ± 4.0% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Co2+: 79 ± 4.0% relative activity, Apo: 4.0 ± 0.33%, and Fe2+: 100 ± 4.6%. Its footnote defines the untreated native preparation separately.

    Source locationPMC4817193 #T2, Co2+, Apo and Fe2+ rows; relative-activity column and footnote
manganese(2+)
CHEBI:29035		activates

In the 2016 metal-substitution assay, Mn2+ supported 30 ± 1.9% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Mn2+: 30 ± 1.9% relative activity, Apo: 4.0 ± 0.33%, and Fe2+: 100 ± 4.6%. Its footnote defines the untreated native preparation separately.

    Source locationPMC4817193 #T2, Mn2+, Apo and Fe2+ rows; relative-activity column and footnote
zinc(2+)
CHEBI:29105		activates

In the 2016 metal-substitution assay, Zn2+ supported 17 ± 0.66% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Zn2+: 17 ± 0.66% relative activity, Apo: 4.0 ± 0.33%, and Fe2+: 100 ± 4.6%. Its footnote defines the untreated native preparation separately.

    Source locationPMC4817193 #T2, Zn2+, Apo and Fe2+ rows; relative-activity column and footnote
iron(3+)
CHEBI:29034		activates

In the 2016 metal-substitution assay, Fe3+ supported 14 ± 2.2% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Fe3+: 14 ± 2.2% relative activity, Apo: 4.0 ± 0.33%, and Fe2+: 100 ± 4.6%. Its footnote defines the untreated native preparation separately.

    Source locationPMC4817193 #T2, Fe3+, Apo and Fe2+ rows; relative-activity column and footnote
nickel(2+)
CHEBI:49786		activates

In the 2016 metal-substitution assay, Ni2+ supported 13 ± 0.33% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Ni2+: 13 ± 0.33% relative activity, Apo: 4.0 ± 0.33%, and Fe2+: 100 ± 4.6%. Its footnote defines the untreated native preparation separately.

    Source locationPMC4817193 #T2, Ni2+, Apo and Fe2+ rows; relative-activity column and footnote
calcium(2+)
CHEBI:29108		activates

In the 2016 metal-substitution assay, Ca2+ supported 11 ± 1.0% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Ca2+: 11 ± 1.0% relative activity, Apo: 4.0 ± 0.33%, and Fe2+: 100 ± 4.6%. Its footnote defines the untreated native preparation separately.

    Source locationPMC4817193 #T2, Ca2+, Apo and Fe2+ rows; relative-activity column and footnote
magnesium(2+)
CHEBI:18420		activates

In the 2016 metal-substitution assay, Mg2+ supported 9.8 ± 0.66% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Mg2+: 9.8 ± 0.66% relative activity, Apo: 4.0 ± 0.33%, and Fe2+: 100 ± 4.6%. Its footnote defines the untreated native preparation separately.

    Source locationPMC4817193 #T2, Mg2+, Apo and Fe2+ rows; relative-activity column and footnote
cadmium(2+)
CHEBI:48775		activates

In the 2016 metal-substitution assay, Cd2+ supported 7.4 ± 0.66% activity relative to Fe2+ = 100%; the apo row is 4.0 ± 0.33%. Assay: 50 µM CHM and 50 µM metal, 100 mM HEPES pH 7.0, 25 °C; apoenzyme received at least 1 h preincubation. Metal stripping used 1 mM EDTA for 12 h at 4 °C followed by washing. Fe2+ was incubated anaerobically on ice; other metal-substitution preparation is described at 15 °C. This is conditional reconstitution, not physiological cofactor exclusivity.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Cd2+: 7.4 ± 0.66% relative activity, Apo: 4.0 ± 0.33%, and Fe2+: 100 ± 4.6%. Its footnote defines the untreated native preparation separately.

    Source locationPMC4817193 #T2, Cd2+, Apo and Fe2+ rows; relative-activity column and footnote
4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
has_cofactor

2016 reconstituted WT GalB supports CHM hydration with Fe2+. Table 4 gives a full saturation series. This is an experimentally usable alternative metal in the specified preparation; aerobic KT2442 purification predominantly retained Zn and does not establish exclusive in vivo occupancy.

iron(2+)
CHEBI:29033
  • PMID:26867578

    Source assertion: Table 4 gives Wild type CHM kinetic rows for Fe2+, Co2+ and Zn2+ at 50 µM metal; Table 2 gives corresponding restored activities.

    Source locationPMC4817193 #T4 Wild type / CHM / metal rows; #T2 corresponding metal rows
4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB from pET29a-derived constructs, overexpressed in E. coli BL21(λDE3) or KT2442/pVLT31; the table does not assign a host per kinetic row. EDTA-stripped apoenzyme was reconstituted before assay. Full insert remains unresolved (2011 244 residues versus reference 258); the 240-residue crystal construct is separate.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names GalB-catalyzed CHM hydration and separately lists native, apo and EDTA preparations; Table 4 labels the unmutated preparation Wild type.

    Source locationPMC4817193 #T2 title/Native/Apo/EDTA rows and #T4 Wild type rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, preincubation sentence
has_cofactor

2016 reconstituted WT GalB supports CHM hydration with Co2+. Table 4 gives a full saturation series. This is an experimentally usable alternative metal in the specified preparation; aerobic KT2442 purification predominantly retained Zn and does not establish exclusive in vivo occupancy.

cobalt(2+)
CHEBI:48828
  • PMID:26867578

    Source assertion: Table 4 gives Wild type CHM kinetic rows for Fe2+, Co2+ and Zn2+ at 50 µM metal; Table 2 gives corresponding restored activities.

    Source locationPMC4817193 #T4 Wild type / CHM / metal rows; #T2 corresponding metal rows
ethylenediaminetetraacetic acid
CHEBI:4735		inhibits

2016 EDTA treatment reduces CHM hydratase activity. Table 2 EDTA row is 0.030 ± 0.0033% relative to Fe2+. Separate Zn-loaded time courses gave half-lives 32.3 min at 250 µM EDTA and 17.1 min at 1 mM; 12 h at 1 mM lowered activity below assay error. The 2011 post-treatment rescue failure used a different protocol and is not overwritten. EDTA named-parent normalization does not establish its supplied salt/protonation.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB in the EDTA-present activity row, separate from the washed apo and metal-supplemented rows. Methods specify 50 µM CHM with 50 µM EDTA for the comparison; separate Zn-loaded time courses used 250/1000 µM EDTA. Complete GalB insert/termini remain unresolved; this is not a reconstituted-metal assay.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 separately lists EDTA, Apo and metal-supplemented GalB activity rows. The EDTA value is 0.030 ± 0.0033%; the Apo value is 4.0 ± 0.33%.

    Source locationPMC4817193 #T2 EDTA/Apo rows and footnote
  • PMID:26867578
    EDTA
    Source locationPMC4817193 #sec16 Enzyme Assays, metal-ion-specificity assay sentence
  • PMID:26867578

    Source assertion: Table 2 lists EDTA: 0.030 ± 0.0033% and Apo: 4.0 ± 0.33% relative activity. These are distinct preparation rows, not universal absence of catalysis.

    Source locationPMC4817193 #T2 EDTA/Apo/Fe2+ rows
cobalt(2+)
CHEBI:48828		activates

In the separately identified KT2442 overexpression/purification preparation, 50 µM CoCl2 for 10 min increased relative CHM activity from 8.6 ± 0.95% to 34 ± 1.5%. This indicates underloading/loss during preparation, not a physiological cobalt preference.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: GalB overexpressed in P. putida KT2442 from pVLT31, aerobically purified by three chromatographic steps; the Table 2 native preparation precedes EDTA stripping. The complete GalB insert is unresolved.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 distinguishes the purified native enzyme before EDTA treatment and its cobalt-supplemented counterpart.

    Source locationPMC4817193 #T2 Native/Native + Co2+ rows and footnote
  • PMID:26867578

    Source assertion: Table 2 lists Native: 8.6 ± 0.95% and Native + Co2+: 34 ± 1.5%; footnote specifies 50 µM CoCl2 for 10 min.

    Source locationPMC4817193 #T2 Native and Native + Co2+ rows and footnote
4-oxalomesaconate hydratase [Comamonas sp. E6]
NCBIProtein:BAI50711.1

experimental construct: Codon-optimized Comamonas E6 pmdE (BAI50711.1) synthesized into pET28a, expressed in E. coli BL21(λDE3), initially N-His6-tagged. Thrombin cleavage and Ni-NTA removal of tag/tagged material were performed; full insert and residual cleavage scar remain unresolved.

Qualification evidence
  • PMID:26867578
    polyhistidine tag
    Source locationPMC4817193 #sec8 Protein Purification, LigJCsE6 tag-removal paragraph
catalyzes

The 2016 Comamonas sp. E6 PmdE/LigJ preparation substitutes for GalB in the GalA–GalD–hydratase–GalC coupled assay, giving 1:1 NADH oxidation relative to added gallate. With GalD omitted, only slow hydratase-independent turnover remains. Polarimetry identifies the same negative CHA enantiomer as GalB, without proving absolute S configuration. The source uses the GalD-generated E,E enol; this exact E6 construct is distinct from the older native PmdE homology annotation and modern SYK-6 keto-KCH reaction.

4-carboxy-2-hydroxy-cis,cis-muconate + H2O => 2-hydroxy-4-oxobutane-1,2,4-tricarboxylate
RHEA:17403
  • PMID:26867578
    utilizes the same CHM isomer as GalB
    Source locationPMC4817193 #sec28 Comparative Analysis of GalB and LigJCsE6, coupled-assay results paragraph
gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli; cited 2009 preparation used BL21(DE3) and POROS PI/HQ. Exact tag/vector termini are not supplied. For solution assays, 20-fold EDTA treatment for 12 h at 4 °C preceded anaerobic reactivation with equal volumes of protein and FeCl2/ascorbate solution.

Qualification evidence
  • PMID:24657997
    overexpressed using Escherichia coli
    Source locationPLOS XML #s2a first paragraph; #s2f supplies reactivation context
catalyzes

Reactivated recombinant DesB depleted gallate at 116.1 ± 0.5 U/mg in air-saturated 50 mM Tris-HCl pH 7.5, 30 °C, correcting for product absorbance and substrate autodegradation. This is a different preparation/assay from 2005. The bound-gallate crystal supports substrate coordination but does not identify the immediate solution product tautomer. The shifted iron-site interpretation is a structural model, not directly observed proton transfer.

3,4,5-trihydroxybenzoate + O2 => (1E)-4-oxobut-1-ene-1,2,4-tricarboxylate + 2 H(+)
RHEA:28928
  • PMID:24657997
    116.1±0.5 U/mg
    Source locationPLOS XML #s3a, WT-versus-His192Phe activity paragraph; #s2f methods
gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli; cited 2009 preparation used BL21(DE3) and POROS PI/HQ. Exact tag/vector termini are not supplied. For solution assays, 20-fold EDTA treatment for 12 h at 4 °C preceded anaerobic reactivation with equal volumes of protein and FeCl2/ascorbate solution.

Qualification evidence
  • PMID:24657997
    overexpressed using Escherichia coli
    Source locationPLOS XML #s2a first paragraph; #s2f supplies reactivation context
has_cofactor

Anaerobic structures place Fe(II) at the intersubunit active sites; solution activity used iron/ascorbate-reconstituted apoenzyme. Static coordination distances and the refined alternative iron positions do not establish a two-iron stoichiometry or a directly observed proton transfer.

iron(2+)
CHEBI:29033
  • PMID:24657997
    non-heme Fe (II) ion
    Source locationPLOS XML #s3a first paragraph; Figure 2B and supporting Tables S2/S3
gallate dioxygenase
MetaCyc:CPLX-7918

experimental construct: 2014 recombinant SYK-6 WT DesB expressed in E. coli, crystallized anaerobically. Exact tag/vector termini are not supplied; the crystal assembly is distinguished from the 2021 DesB-MBP fusion.

Qualification evidence
  • PMID:24657997
    overexpressed using Escherichia coli
    Source locationPLOS XML #s2a first paragraph; #s2f supplies reactivation context
has_part

The 2014 crystal is a swapped homodimer: residues 1–300 of one subunit form a protomer with residues 301–C terminus of the other. Active sites lie at the interface. This is not the separate LigAB alpha/beta assembly and does not prove that an isolated DesB monomer is catalytic.

gallate dioxygenase monomer
MetaCyc:MONOMER-15119
  • PMID:24657997
    swap dimer
    Source locationPLOS XML #s3a first paragraph and Figure 2A
Gallate transporter
UniProtKB:E8ZB61

experimental construct: Repaired 449-residue KTGAL GalT expressed from multicopy pIZGalT in the cited whole-cell uptake experiments. This does not identify the inactive KT2440 frameshift or the unsequenced 2023 genomic insert with repaired GalT.

Qualification evidence
  • PMID:21219457
    multicopy plasmid pIZGalT
    Source location2011 Results GalT, publisher line224
enables

The broad activity label is scoped here to repaired KTGAL GalT: gallate uptake and less efficient protocatechuate uptake in whole cells. The 449-residue allele is distinct from the inactive KT2440 frameshift annotation. Gallate/PCA Vmax values 8.4/3.2 nmol/min/mg and Km 4.3/9.1 µM are reported; the adjacent prose emphasizes pIZGalT whereas Figure 2B caption names GA-induced KTGAL, so the complete kinetic series is not uniformly reassigned to the multicopy construct. Tested 50 µM negatives remain conditional. No proton stoichiometry or direct ligand-binding measurement is inferred.

carboxylic acid transmembrane transporter activity
GO:0046943
  • UniProtKB:E8ZB61

    Source assertion: FUNCTION assigns gallate uptake with ECO:0000269/PMID21219457; CAUTION distinguishes the functional sequence from the KT2440 frameshift. GO0046943 is separately an IEA:TreeGrafter annotation.

    Source location$.comments[commentType=FUNCTION or CAUTION]; $.uniProtKBCrossReferences[database=GO,id=GO:0046943]
  • PMID:21219457
    protocatechuate
    Source location2011 Results GalT, publisher line225; Figure2A-B context
Gallate transporter
UniProtKB:E8ZB61		located_in

Plasma-membrane location is an IEA:TreeGrafter annotation; the separate UniProt membrane/multipass comment has ECO:0000305. Neither is described as a directly imaged localization experiment.

plasma membrane
GO:0005886
  • UniProtKB:E8ZB61

    Source assertion: The GO cross-reference GO0005886 has GoTerm C:plasma membrane and GoEvidenceType IEA:TreeGrafter.

    Source location$.uniProtKBCrossReferences[database=GO,id=GO:0005886].properties
Porin-like protein GalP
UniProtKB:Q88JX6		enables

Porin activity is inferred from the IEA:InterPro annotation and Opr-family similarity. In the 2011 KTGAL galP disruption, 10 µM gallate uptake fell from 7.4 to 4.2 nmol/min/mg while growth on gallate was unaffected; this is genetic evidence of a low-concentration uptake contribution, not a purified porin flux or binding assay.

porin activity
GO:0015288
  • UniProtKB:Q88JX6

    Source assertion: The GO0015288 cross-reference is F:porin activity with IEA:InterPro; FUNCTION is a probable low-gallate uptake role (ECO:0000305).

    Source location$.uniProtKBCrossReferences[database=GO,id=GO:0015288].properties; $.comments[commentType=FUNCTION]
  • PMID:21219457
    decreased to half
    Source location2011 Results galP paragraph publisher line229
Porin-like protein GalP
UniProtKB:Q88JX6		located_in

Membrane is the issuing GO annotation (IEA:InterPro). An outer-membrane role is inferred from Opr-family similarity; direct localization and purified transport were not established in the primary uptake study.

membrane
GO:0016020
  • UniProtKB:Q88JX6

    Source assertion: The GO0016020 cross-reference is C:membrane with IEA:InterPro. SIMILARITY assigns the outer membrane porin Opr family with ECO:0000305.

    Source location$.uniProtKBCrossReferences[database=GO,id=GO:0016020].properties; $.comments[commentType=SIMILARITY]
carboxylic acid transmembrane transporter activity
GO:0046943		part_of

Ontology-level activity-to-process context. Both terms remain broad; this does not redefine either GO class as gallate-specific.

carboxylic acid transmembrane transport
GO:1905039
  • GO:0046943

    Source assertion: GO0046943 has an rdfs:subClassOf restriction with owl:onProperty BFO0000050 (part of) and owl:someValuesFrom GO1905039.

    Source locationPinned GO OWL/RDF: GO_0046943 rdfs:subClassOf _:riog00237193; _:riog00237193 owl:onProperty BFO_0000050 and owl:someValuesFrom GO_1905039
carbonyl cyanide p-trifluoromethoxyphenylhydrazone
CHEBI:75458		inhibits

Only the measured gallate uptake instance of this broad transport process is implicated: 30 µM FCCP inhibited whole-cell uptake of 50 µM gallate by KTGAL carrying pIZGalT. This supports proton-motive-force dependence, not direct GalT binding, inhibition of every carboxylate transport process, or a proton stoichiometry. Figure 2C caption gives µM FCCP/CCCP; a PDF text extraction reading mM is rejected.

carboxylic acid transmembrane transport
GO:1905039
  • PMID:21219457
    inhibited
    Source location2011 publisher line226; Figure2C caption lines221–222 specifies each dose and cell context
CCCP
CHEBI:3259		inhibits

Only the measured gallate uptake instance of this broad transport process is implicated: 20 µM CCCP inhibited whole-cell uptake of 50 µM gallate by KTGAL carrying pIZGalT. This supports proton-motive-force dependence, not direct GalT binding, inhibition of every carboxylate transport process, or a proton stoichiometry. Figure 2C caption gives µM FCCP/CCCP; a PDF text extraction reading mM is rejected.

carboxylic acid transmembrane transport
GO:1905039
  • PMID:21219457
    inhibited
    Source location2011 publisher line226; Figure2C caption lines221–222 specifies each dose and cell context
2,4-dinitrophenol
CHEBI:42017		inhibits

Only the measured gallate uptake instance of this broad transport process is implicated: 1 mM DNP inhibited whole-cell uptake of 50 µM gallate by KTGAL carrying pIZGalT. This supports proton-motive-force dependence, not direct GalT binding, inhibition of every carboxylate transport process, or a proton stoichiometry. Figure 2C caption gives µM FCCP/CCCP; a PDF text extraction reading mM is rejected.

carboxylic acid transmembrane transport
GO:1905039
  • PMID:21219457
    inhibited
    Source location2011 publisher line226; Figure2C caption lines221–222 specifies each dose and cell context
diethyl pyrocarbonate
CHEBI:59051		inhibits

Diethyl pyrocarbonate at 0.5 mM for 7 min at 25 °C reduced purified GalB hydratase activity to 15% of untreated. The result suggests an important histidine but does not identify the modified residue or prove a specific proton-transfer mechanism.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2011 purified GalB from E. coli BL21(DE3)/pETGalB, purified by DEAE and phenyl chromatography. Table 1 reports 244 residues/27.4 kDa; complete insert and termini are unresolved against the 258-residue deposited reference. DEPC assay: 0.5 mM, 7 min, 25 °C.

Qualification evidence
  • MetaCyc:ENZRXN-18373

    Source assertion: The activity has purified heterologous-host evidence for CPLX-8178 linked to PMID21219457.

    Source location/ptools-xml/Enzymatic-Reaction/evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]
  • PMID:21219457

    Source assertion: Table 1 lists GalB as a 244-residue, 27.4-kDa protein in the gal cluster. This does not identify the complete recombinant insert with the independently deposited 258-residue reference.

    Source location2011 Table1, galB row: protein size and predicted molecular mass columns
  • PMID:21219457
    pETGalB
    Source location2011 Results GalB publisher line274: E. coli BL21(DE3)(pETGalB); Methods GalB expression and purification
  • PMID:21219457
    to 15%
    Source location2011 Results GalB, publisher line278; Methods protein modification conditions
ethylenediaminetetraacetic acid
CHEBI:4735		inhibits

The 2011 experiment reports concentration-dependent inhibition after EDTA treatment (3 h, 4 °C) and no restoration after the tested post-treatment metal additions. This protocol is distinct from the extensively washed/reconstituted 2016 apoenzyme; the two results are not flattened into a universal rescue claim. EDTA is a named-parent normalization with unspecified supplied salt/protonation.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2011 E. coli BL21(DE3)/pETGalB cell extract in the EDTA hydratase experiments. Table 1 reports 244 residues/27.4 kDa; complete insert/termini remain unresolved against the 258-residue reference. This extract assay is separate from purified GalB modification and 2016 washed apoenzyme.

Qualification evidence
  • MetaCyc:ENZRXN-18373

    Source assertion: The activity has purified heterologous-host evidence for CPLX-8178 linked to PMID21219457.

    Source location/ptools-xml/Enzymatic-Reaction/evidence[Evidence-Code/@frameid="EV-EXP-IDA-PURIFIED-PROTEIN-HH"]
  • PMID:21219457

    Source assertion: Table 1 lists GalB as a 244-residue, 27.4-kDa protein in the gal cluster. This does not identify the complete recombinant insert with the independently deposited 258-residue reference.

    Source location2011 Table1, galB row: protein size and predicted molecular mass columns
  • PMID:21219457
    pETGalB
    Source location2011 Results GalB publisher line274: E. coli BL21(DE3)(pETGalB); Methods GalB expression and purification
  • PMID:21219457
    inhibited
    Source location2011 Results GalB publisher lines274–275 and Figure6 caption/protocol
gallate
CHEBI:16918		regulates

In citrate-grown KT2440, adding 1 mM gallate for 2 h induced the galA RT-PCR signal in the tested aromatic panel. This is indirect whole-cell transcriptional response, not direct gallate–GalR binding or GalA protein activation. The 2005 insertional growth mutant and the later KTGAL repaired transporter strain are separate experimental contexts.

PP_2518
KEGG:ppu:PP_2518
  • PMID:16030014
    specifically induced
    Source locationGalA2005 abstract transcript lines172–176; Results and Figure6 caption lines1800–1846; methods lines923–955
gallate
CHEBI:16918		regulates

The 2011 KTGAL RT-PCR study reports gallate-inducible galBCD and galTAP transcriptional units; citrate-only cultures lacked the tested intergenic products. This edge denotes an indirect environmental transcriptional response at the stated promoter/gene context. Insertions in galB and galT caused downstream polar effects, so individual-gene necessity is not isolated. The gene locus owner is distinct from the repaired KTGAL GalT protein; no direct GalR ligand binding is established.

PP_2515
KEGG:ppu:PP_2515
  • PMID:21219457
    GA inducible
    Source location2011 Transcriptional organization, publisher lines236–239; FigureS2 original unavailable
gallate
CHEBI:16918		regulates

The 2011 KTGAL RT-PCR study reports gallate-inducible galBCD and galTAP transcriptional units; citrate-only cultures lacked the tested intergenic products. This edge denotes an indirect environmental transcriptional response at the stated promoter/gene context. Insertions in galB and galT caused downstream polar effects, so individual-gene necessity is not isolated. The gene locus owner is distinct from the repaired KTGAL GalT protein; no direct GalR ligand binding is established.

PP_2519
KEGG:ppu:PP_2519
  • PMID:21219457
    GA inducible
    Source location2011 Transcriptional organization, publisher lines236–239; FigureS2 original unavailable
copper(2+)
CHEBI:29036		inhibits

Under the 2016 metal-substitution assay, Cu2+ gave 0.19 ± 0.10% CHM hydration activity versus 4.0 ± 0.33% in the apo row, with Fe2+ = 100%. This is lower activity in a 50 µM Cu2+-challenged apo preparation (50 µM CHM, 100 mM HEPES pH 7.0, 25 °C), not a measured physiological inhibition or an untreated Zn-loaded GalB effect. Metal stripping/washing and preincubation conditions are distinct from the EDTA-present row.

4-oxalmesaconate hydratase
MetaCyc:CPLX-8178

experimental construct: 2016 WT GalB apo preparation after EDTA stripping and washing, incubated with 50 µM Cu2+ before the CHM assay. Host is not assigned per table row; full insert/termini remain unresolved (2011 244 residues versus reference 258). This is distinct from the separately measured EDTA-present preparation.

Qualification evidence
  • PMID:26867578

    Source assertion: Table 2 names the Cu2+ and Apo preparations separately.

    Source locationPMC4817193 #T2 Cu2+/Apo rows
  • PMID:26867578
    apoGalB
    Source locationPMC4817193 #sec16 Enzyme Assays, metal-preincubation sentence
  • PMID:26867578

    Source assertion: Table 2 lists Cu2+: 0.19 ± 0.10% and Apo: 4.0 ± 0.33% relative activity, with Fe2+ set to 100 ± 4.6%.

    Source locationPMC4817193 #T2 Cu2+/Apo/Fe2+ rows; relative-activity column
4-oxalmesaconate hydratase monomer
MetaCyc:G1G01-2699-MONOMER		located_in

The native KT2440 GalB monomer has an explicit cytosol location and a matching GO annotation supported by EV-COMP-AINF with PSORTdb (PMID:26602691). This preserves a computational annotation, not a direct localization assay. It does not assign localization to the separately qualified 2011 or 2016 recombinant preparations or to every pathway component.

cytosol
GO:0005829
  • MetaCyc:G1G01-2699-MONOMER

    Source assertion: The Protein object assigns location CCO-CYTOSOL. Its sixth has-go-term slot is GO:0005829; both evidence entries use EV-COMP-AINF and PUB-26602691 (PSORTdb).

    Source location/ptools-xml/Protein[@frameid='G1G01-2699-MONOMER']/location/cco; /ptools-xml/Protein[@frameid='G1G01-2699-MONOMER']/has-go-term[6]/GO-Term; has-go-term[6]/evidence; original lines 128–140 and 203

References

Curation history