PA0949 is the Pseudomonas aeruginosa ortholog of WrbA, a soluble flavodoxin-like FMN-containing NAD(P)H:quinone oxidoreductase (type IV NQO, EC 1.6.5.2). Crystal structures show a long-chain flavodoxin fold with the WrbA-specific insertion, one non-covalently bound FMN per protomer, and a homotetrameric assembly with 222 symmetry. The purified P. aeruginosa enzyme reduces quinones by a concerted two-electron transfer with minimal semiquinone formation, which avoids reactive semiquinone radicals. Over-expression of PA0949 improves growth under H2O2 challenge in a catalase (katA) mutant and in P. putida, but not in wild-type P. aeruginosa, so a physiological antioxidant role is plausible but not established.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003955 NAD(P)H dehydrogenase (quinone) activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the PAINT IBD on PTN000777387 (WrbA family). Seeds are E. coli WrbA (IDA/IMP, PMID:16672604), yeast PST2 (IDA), Arabidopsis FQR1 (IDA) and the PseudoCAP IMP on PA0949 itself, which is expected and not circular. The IBD stands without the PA0949 seed and suits a WrbA-family clade. Reason: Core activity of the WrbA family, directly shown for the purified P. aeruginosa enzyme and for three independent seeds. Supporting Evidence: PMID:25085734 In in vitro assays, each enzyme was shown to reduce quinone substrates with only minimal semiquinone formation. PMID:16672604 suggesting that this activity is a defining characteristic of the WrbA family that we designate a new type of NQO (type IV) |
| GO:0003955 NAD(P)H dehydrogenase (quinone) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic mapping from the WrbA-like InterPro family and EC 1.6.5.2. Reason: Consistent with the IBA and the direct assay on the P. aeruginosa enzyme. Supporting Evidence: PMID:25085734 In in vitro assays, each enzyme was shown to reduce quinone substrates with only minimal semiquinone formation. |
| GO:0003955 NAD(P)H dehydrogenase (quinone) activity | IMP PMID:25085734 Pseudomonas aeruginosa MdaB and WrbA are water-soluble two-e... | ACCEPT | Summary: PseudoCAP seed row for the PTN000777387 IBD. The abstract states that the purified P. aeruginosa WrbA ortholog reduces quinones in vitro, which is direct evidence for this activity (an IDA-type observation coded as IMP). Reason: Term is correct and supported by the paper for this gene product; seed verdict SOUND. The evidence code would more naturally be IDA, but this does not change the claim (abstract only, so not filed as a defect). Supporting Evidence: PMID:25085734 In in vitro assays, each enzyme was shown to reduce quinone substrates with only minimal semiquinone formation. |
| GO:0008753 NADPH dehydrogenase (quinone) activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea mapping of the NADPH reaction from the UniProt catalytic activity (by similarity to E. coli WrbA). Reason: WrbA-family enzymes use both NADH and NADPH; the E. coli ortholog has experimental support for both. This specific descendant of GO:0003955 is reasonable for PA0949. Supporting Evidence: PMID:16672604 WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase. |
| GO:0008753 NADPH dehydrogenase (quinone) activity | ISS GO_REF:0000024 | ACCEPT | Summary: ISS from E. coli WrbA (P0A8G6). Reason: Same claim as the Rhea row; consistent action. Supporting Evidence: PMID:16672604 WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase. |
| GO:0009055 electron transfer activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO from the flavodoxin signature. Flavodoxins are one-electron carriers; WrbA is an enzyme that reduces quinones by two-electron hydride transfer and is not known to shuttle electrons between redox partners. Reason: The flavodoxin-fold mapping assigns electron-carrier activity, which is not what WrbA-family proteins do; their activity is captured by GO:0003955. Supporting Evidence: PMID:25085734 Water-soluble quinone oxidoreductases capable of reducing quinone substrates via a concerted two-electron mechanism |
| GO:0010181 FMN binding | IEA GO_REF:0000002 | ACCEPT | Summary: FMN cofactor binding, seen directly in the P. aeruginosa crystal structure. Reason: Supported by crystal structures with bound FMN. Supporting Evidence: PMID:16322580 each WrbA protein forms a homotetramer with 222 symmetry, unique among flavodoxin-like proteins, in which each protomer binds one flavin mononucleotide cofactor molecule. |
| GO:0016020 membrane | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from PTN000777387; all seeds are fungal flavodoxin-like proteins (yeast YCP4, RFS1; Candida PST1-3, YCP4) annotated to plasma membrane or membrane raft. PA0949 has no TM segment and is described as a water-soluble enzyme; no bacterial member contributes membrane evidence. Reason: The membrane association of the fungal Pst/Ycp4/Rfs1 proteins is a lineage-specific feature not shown for bacterial WrbA. PA0949 is a soluble cytoplasmic quinone reductase by all evidence available. Not filed as a GO_CENTRAL defect because PA0949 is not a seed for this term and the title-level evidence ("water-soluble") does not exclude peripheral association; raised as a question instead. Propagation Review Root cause: PROPAGATION BAD Failure modes: LINEAGE OR TAXON MISMATCH COMPARTMENT OR COMPLEX MISMATCH Supporting Evidence: PMID:25085734 Pseudomonas aeruginosa MdaB and WrbA are water-soluble two-electron quinone oxidoreductases |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: Generic InterPro2GO term. Reason: Entailed by GO:0003955, accepted above. |
| GO:0016655 oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor | IDA PMID:16672604 WrbA from Escherichia coli and Archaeoglobus fulgidus is an ... | TOO GENERAL | Summary: PseudoCAP IDA citing the E. coli/A. fulgidus WrbA paper. Full text read: the paper assays only the E. coli and A. fulgidus proteins and mentions P. aeruginosa WrbA only as a published crystal structure. The term is true of PA0949 (direct assay in PMID:25085734) but the IDA is attached to the wrong paper for this gene product. Reason: The claim is true but entailed by GO:0003955 (is_a GO:0016655), accepted above. Separately, the row's reference does not assay PA0949; a source defect is recorded. Supporting Evidence: PMID:25085734 In in vitro assays, each enzyme was shown to reduce quinone substrates with only minimal semiquinone formation. |
| GO:0034599 cellular response to oxidative stress | IMP PMID:25085734 Pseudomonas aeruginosa MdaB and WrbA are water-soluble two-e... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IMP. The abstract reports no H2O2-protective phenotype for over-expression in wild-type P. aeruginosa; protection is seen only in a katA mutant or in P. putida, and no loss-of-function phenotype is reported. The authors themselves call this a potential role. Reason: Over-expression in a sensitised background shows capacity, not participation in the native response. Plausible but not established for PA0949 (abstract only; a wrong-row defect is not filed). This row does not seed an IBD. Supporting Evidence: PMID:25085734 However, when each was individually over-expressed in P. aeruginosa no overt H2O2-protective phenotype was observed. |
| GO:0050136 NADH dehydrogenase (quinone) (non-electrogenic) activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea mapping of the NADH reaction (by similarity to E. coli WrbA). Reason: Soluble WrbA NADH:quinone reduction is non-electrogenic; E. coli WrbA has experimental support. Specific descendant of GO:0003955. Supporting Evidence: PMID:16672604 For E. coli WrbA, the K(m)(NADH) was 14 +/- 0.43 microM |
| GO:0050136 NADH dehydrogenase (quinone) (non-electrogenic) activity | ISS GO_REF:0000024 | ACCEPT | Summary: ISS from E. coli WrbA (P0A8G6). Reason: Same claim as the Rhea row; consistent action. Supporting Evidence: PMID:16672604 For E. coli WrbA, the K(m)(NADH) was 14 +/- 0.43 microM |
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Download this section (compressed HTML)Q: The PTN000777387 membrane IBA (GO:0016020) rests only on fungal Pst/Ycp4/Rfs1 seeds and propagates to bacterial WrbA proteins such as PA0949 and E. coli WrbA. Should the IBD be moved to a fungal subclade node?
Q: Does loss of PA0949 (alone or with mdaB) sensitise P. aeruginosa to quinone or H2O2 stress, and what is the physiological quinone substrate?
Experiment: Test a PA0949 deletion (in wild-type and katA backgrounds) for sensitivity to menadione and H2O2, and fractionate cells to confirm the soluble cytoplasmic location.
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