pcaG encodes the alpha subunit of the heteromeric protocatechuate 3,4-dioxygenase (PcaHG) in Pseudomonas putida KT2440. In the assembled PcaHG complex, the alpha chain helps form the catalytic interface that carries out Fe(III)-dependent intradiol ring cleavage of protocatechuate to 3-carboxy-cis,cis-muconate in the protocatechuate branch of the beta-ketoadipate pathway. KT2440 deletion experiments show that loss of pcaHG blocks downstream protocatechuate consumption, while structural studies of the Pseudomonas enzyme indicate that ferric-iron ligation is provided by beta-subunit residues rather than by pcaG itself.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Technically true but much too generic to be useful once the specific protocatechuate 3,4-dioxygenase term is present. Reason: GO:0018578 captures the relevant biochemical activity and substrate specificity. Retaining GO:0003824 does not add meaningful functional resolution. |
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | REMOVE | Summary: Unsupported for the pcaG alpha subunit. Structural analyses place the catalytic Fe3+ ligands on beta-subunit residues, not on pcaG. Reason: The alpha chain contributes to the catalytic interface, but the iron-ligating residues defined for protocatechuate 3,4-dioxygenase are all on the beta subunit. This makes the iron-binding annotation a subunit-level overassignment for pcaG. Falcon deep research independently describes the catalytic Fe(III) as a 2-His/2-Tyr center, a holoenzyme-level cofactor property rather than an alpha-subunit function. Supporting Evidence: PMID:9485360 is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta) PMID:9254600 Tyr447 is stabilized by hydrogen bonding to Tyr16 (16alpha) and Asp413 (113beta) file:PSEPK/pcaG/pcaG-deep-research-falcon.md the catalytic metal is **Fe(III)** coordinated by a **2-His/2-Tyr ligand set**, and crystallography captured **alkylperoxo and anhydride intermediates** following O2 addition |
| GO:0008199 ferric iron binding | IEA GO_REF:0000002 | REMOVE | Summary: Also unsupported for pcaG specifically. The holoenzyme uses Fe3+, but the identified ferric-iron ligands belong to the beta subunit. Reason: This annotation confuses a complex-level cofactor requirement with the subunit-level molecular function of pcaG. The structural data support retaining the complex dioxygenase activity while dropping ferric-iron binding on the alpha chain. Falcon deep research reinforces this by describing the Fe(III) 2-His/2-Tyr center as a property of the assembled 3,4-PCD enzyme. Supporting Evidence: PMID:9485360 is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta) PMID:9254600 Tyr447 is stabilized by hydrogen bonding to Tyr16 (16alpha) and Asp413 (113beta) file:PSEPK/pcaG/pcaG-deep-research-falcon.md the catalytic metal is **Fe(III)** coordinated by a **2-His/2-Tyr ligand set**, and crystallography captured **alkylperoxo and anhydride intermediates** following O2 addition |
| GO:0016702 oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Broad parent term that is consistent with dioxygenase chemistry but over-annotated relative to the specific protocatechuate 3,4-dioxygenase term. Reason: The specific molecular function GO:0018578 already captures the relevant oxygen-incorporating chemistry and substrate identity. The parent oxidoreductase term is therefore redundant. |
| GO:0018578 protocatechuate 3,4-dioxygenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Correct core molecular function. pcaG is the alpha chain of the PcaHG protocatechuate 3,4-dioxygenase that cleaves protocatechuate in aromatic catabolism. Reason: Classical cloning and biochemical work identify pcaG as the alpha subunit of protocatechuate 3,4-dioxygenase, Pseudomonas enzyme biochemistry shows an alpha-beta catalytic unit architecture, and KT2440 deletion experiments confirm the same two-subunit enzyme assignment in this strain. The qualifier should be contributes_to because the activity is realized by the assembled PcaHG complex, not by the isolated alpha chain. Supporting Evidence: PMID:8407791 contain two successive open reading frames, designated pcaH and pcaG, corresponding to the beta and alpha subunits, respectively, of 3,4-PCD PMID:6273403 contains 4 alpha subunits of 23,000 daltons, 4 beta subunits of 26,500 daltons, and 4 ferric irons suggesting that the enzyme is a tetramer of (alpha beta Fe+3) catalytic units PMID:29765865 deleting the genes pcaH and pcaG, which encode the two-subunit protocatechuate 3,4-dioxygenase file:PSEPK/pcaG/pcaG-deep-research-falcon.md encodes the **Ξ±-subunit of protocatechuate 3,4-dioxygenase (PcaGH; EC 1.13.11.3)**, the canonical intradiol ring-cleaving dioxygenase of the protocatechuate (PCA) branch of the **Ξ²-ketoadipate pathway** |
| GO:0019618 protocatechuate catabolic process, ortho-cleavage | IC PMID:29765865 A protocatechuate biosensor for Pseudomonas putida KT2440 vi... | NEW | Summary: Missing core biological-process term. pcaG contributes to the ortho-cleavage step that commits protocatechuate to downstream beta-ketoadipate metabolism. Reason: KT2440 pcaHG deletion blocks protocatechuate consumption, while classical Pseudomonas pcaGH studies define the enzyme as a protocatechuate ortho-cleavage dioxygenase. This term captures the direct pathway step more precisely than a generic aromatic catabolism term. Supporting Evidence: PMID:29765865 deleting the genes pcaH and pcaG, which encode the two-subunit protocatechuate 3,4-dioxygenase PMID:10049847 vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 file:PSEPK/pcaG/pcaG-deep-research-falcon.md **ΞpcaGH** blocks PCA ring cleavage, allowing PCA accumulation and preventing further catabolism through the native Ξ²-ketoadipate pathway |
| GO:0042952 beta-ketoadipate pathway | IC PMID:18156252 The target for the Pseudomonas putida Crc global regulator i... | NEW | Summary: Appropriate broader pathway annotation for an enzyme in the central protocatechuate branch of aromatic assimilation. Reason: pcaG is not merely associated with aromatic metabolism in general; it encodes one of the core ring-cleavage subunits that feeds the beta-ketoadipate pathway. This is broader than GO:0019618, but still directly applicable to the enzyme itself. Supporting Evidence: PMID:18156252 the cat and pca genes of the central catechol and beta-ketoadipate pathways PMID:29765865 native (in which PCA is metabolized via the Ξ²-ketoadipate pathway) file:PSEPK/pcaG/pcaG-deep-research-openai.md The pcaG gene product is a central enzyme in the Ξ²-ketoadipate pathway file:PSEPK/pcaG/pcaG-deep-research-falcon.md **pcaGH is the commitment step that prevents PCA accumulation**, because once PCA is cleaved to carboxymuconate, flux is directed into the central Ξ²-ketoadipate pathway |
Loading supporting contentβ¦
Download this section (compressed HTML)Q: Which alpha-subunit interface residues in KT2440 PcaG most strongly determine substrate positioning and turnover once the beta-subunit Fe3+ center is assembled?
Suggested experts: Structural enzymologists, Aromatic-catabolism biochemists
Q: Under mixed lignin-derived aromatic substrates, is PcaHG flux-limiting relative to upstream protocatechuate-producing enzymes in KT2440?
Suggested experts: Pseudomonas systems biologists, Metabolic engineers
Experiment: Mutate alpha-subunit interface residues such as Tyr16 and assay protocatechuate cleavage kinetics, substrate range, and growth on protocatechuate, p-hydroxybenzoate, and vanillate.
Type: Site-directed mutagenesis
Experiment: Compare WT, delta-pcaG, delta-pcaH, and complemented strains during growth on protocatechuate and upstream aromatics to quantify protocatechuate accumulation and downstream beta-ketoadipate intermediates.
Type: Knockout-complement metabolomics
Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)