PA2077 is the 10S-dioxygenase (10S-DOX) of the oleate-diol synthase pathway of Pseudomonas aeruginosa, which converts oleic acid into hydroxylated fatty acids (oxylipins). It oxygenates oleic acid to the hydroperoxide 10-H(P)OME, which its operon partner PA2078 converts to 7,10-DiHOME. PA2077 is a periplasmic di-heme protein (two CXXCH heme c motifs, signal peptide) related to bacterial di-heme cytochrome c peroxidases and the rubber oxygenase RoxA; both hemes are required for its activity. With PA2078 it defines a fatty-acid-acting subfamily (FadCcp) that arose by gene duplication within Pseudomonas. Over-expression of PA2077 without PA2078 is toxic, consistent with accumulation of its hydroperoxide product.
Definition: Catalysis of the reaction oleate + O2 = (10S)-hydroperoxy-(8E)-octadecenoate.
Justification: PA2077 performs this reaction; GO has only the general dioxygenase activity and analogous fatty acid terms such as linoleate 8R-lipoxygenase activity.
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004130 cytochrome-c peroxidase activity | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from the family-level Ccp node PTN000779264, seeded by P. aeruginosa CcpA (P14532) and E. coli YhjA (P37197). PA2077 is a di-heme Ccp-family protein, but its characterized activity is fatty acid dioxygenation, and it falls outside the branch of characterized peroxidases. Reason: Ancestral family activity that has never been tested for PA2077, which has neofunctionalized into an oleate 10S-dioxygenase (FadCcp subfamily). Not shown to be wrong, but not supported for this protein. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000779264 · PTN000779264 SUPPORTS SOURCE BUT NOT TARGET Family-level di-heme Ccp node covering MauG, RoxA-like and FadCcp members; sound for the classical peroxidases but PA2077 has a different, characterized activity. UniProtKB:P14532 · ccpA (P. aeruginosa cytochrome c551 peroxidase) SUPPORTS SOURCE BUT NOT TARGET Classical bacterial Ccp in the main peroxidase branch. UniProtKB:P37197 · yhjA (E. coli) SUPPORTS SOURCE BUT NOT TARGET Classical bacterial Ccp in the main peroxidase branch. Supporting Evidence: PMID:26154497 Nevertheless, the phylogenetic results obtained here indicate that PA2077 and PA2078 do not group in the main phylogenetic branch described so far for functionally characterized Ccps, constituting thus a new cluster of functional and structural enzymes. |
| GO:0009055 electron transfer activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO from the cytochrome c-like domain superfamily. The hemes of PA2077 serve its own catalysis; it is not known to shuttle electrons between partners. Reason: Electron transfer activity describes a carrier; PA2077 is an enzyme whose hemes are catalytic cofactors. Domain-based over-annotation. Supporting Evidence: PMID:26154497 Mutants at either position H130 or H375 (residue numbering with signal peptide) in protein PA2077 failed to convert oleic acid into 10-H(P)OME (Fig 5), definitely showing that both histidines (the two hemes) are required by PA2077 to display activity. |
| GO:0020037 heme binding | IEA GO_REF:0000002 | ACCEPT | Summary: Two CXXCH heme c attachment motifs; mutation of either heme-ligating histidine abolishes activity. Reason: Supported by sequence and by heme-site mutagenesis; the hemes are the catalytic cofactors. Supporting Evidence: PMID:26154497 Mutants at either position H130 or H375 (residue numbering with signal peptide) in protein PA2077 failed to convert oleic acid into 10-H(P)OME (Fig 5), definitely showing that both histidines (the two hemes) are required by PA2077 to display activity. |
| GO:0031408 oxylipin biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN002826263, seeded by PA2077 itself and PA2078. True for PA2077, which has its own IMP evidence. The node also covers PA0007, PA1941, two Bradyrhizobium and one Rhodopirellula protein with no evidence of fatty acid oxygenation. Reason: Correct for this leaf. The node-level problem, a pathway-specific term placed above the PA2077/PA2078 duplication, is recorded here once as a GO_CENTRAL defect; PA2078 cross-references it. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002826263 · PTN002826263 SUPPORTS SOURCE BUT NOT TARGET Sound for PA2077 and PA2078, but the 7-leaf clade includes PA0007, PA1941 and Bradyrhizobium and Rhodopirellula proteins that have not been characterized; the authors describe the pair as Pseudomonas in-paralogs (GO_CENTRAL defect on this row). UniProtKB:Q9I238 · PA2077 (self; PseudoCAP IMP PMID:24980978) SUPPORTS TRANSFER Expected self-seed reflecting the target's own mutant and complementation evidence. UniProtKB:Q9I237 · PA2078 (PseudoCAP IMP PMID:24980978) SUPPORTS TRANSFER Sound seed; PA2078 performs the second step. Supporting Evidence: PMID:24980978 gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction |
| GO:0031408 oxylipin biosynthetic process | IMP PMID:24980978 Unveiling the genes responsible for the unique Pseudomonas a... | ACCEPT | Summary: PseudoCAP IMP and a seed of the PTN002826263 IBD. The PA2077 mutant cannot release hydroxylated fatty acids; complementation assigns it the first, dioxygenase step. Reason: PA2077 catalyses a step of the oxylipin (7,10-DiHOME) synthesis pathway. Seed verdict SOUND. Supporting Evidence: PMID:24980978 gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction |
| GO:0051213 dioxygenase activity | IMP PMID:24980978 Unveiling the genes responsible for the unique Pseudomonas a... | ACCEPT | Summary: PseudoCAP IMP. Complementation and separate heterologous expression show PA2077 is the oleate 10S-dioxygenase producing 10-H(P)OME. Reason: Core activity. GO has no oleate 10S-dioxygenase term, so dioxygenase activity is as precise as the ontology allows; a new term is proposed. Supporting Evidence: PMID:24980978 gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction PMID:24980978 Heterologous expression of both enzymes separately showed that no hetero-complex formation is required for enzymatic activity. |
| GO:0098869 cellular oxidant detoxification | IEA GO_REF:0000108 | REMOVE | Summary: Logical inference from the cytochrome-c peroxidase IBA. PA2077 produces an organic hydroperoxide; its over-expression without PA2078 is toxic. Reason: The inference rests on an untested peroxidase activity, and the measured activity runs the other way: PA2077 generates the hydroperoxide that PA2078 removes. Supporting Evidence: PMID:26154497 Normal growth rates and full activity were achieved in both mutants, indicating that when the two enzymes are co-expressed in trans, both maintain their respective functions, with PA2078 abolishing toxicity. |
| GO:0042597 periplasmic space | IC PMID:26154497 In Silico/In Vivo Insights into the Functional and Evolution... | NEW | Summary: Proposed location. PA2077 has a type I signal peptide (SignalP 1-21) and PhoA fusion screening confirmed export to the periplasm. Reason: Two independent lines agree (signal peptide and PhoA fusion), and the oleate-diol synthase activity is periplasmic; no cellular component is annotated at present. Supporting Evidence: PMID:26154497 both proteins display type I signal export sequences that allow transport of the synthesized products to the periplasm, a fact experimentally confirmed by PhoA fusion screening |
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Download this section (compressed HTML)Q: Do PA0007 and PA1941, the other PAO1 members of PANTHER subfamily PTHR30600:SF9, oxygenate fatty acids, or are they RoxA-like oxygenases with other substrates?
Experiment: Express PA0007 and PA1941 in a PA2077/PA2078 double mutant and test oleic acid and 10-H(P)OME conversion by LC-MS, to decide how far the oxylipin function extends in the subfamily.
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