azoR2 (PA1962) encodes paAzoR2, one of three FMN-dependent flavodoxin-fold azoreductases (azoreductase type 1 family) of Pseudomonas aeruginosa. The purified enzyme uses NADH to reduce quinones and reduces both benzoquinones and naphthoquinones efficiently, consistent with a larger active site than its paralogs; it also reduces large azo dyes such as Ponceau BS and amaranth, at much lower rates. Its likely physiological role, inferred from the Escherichia coli ortholog AzoR, is two-electron reduction of toxic quinones, but no P. aeruginosa mutant phenotype has been reported.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000117 | TOO GENERAL | Summary: True but uninformative ARBA prediction. Reason: GO:0003955 NAD(P)H dehydrogenase (quinone) activity, asserted as a NEW row in this review, is_a catalytic activity. Supporting Evidence: PMID:24915188 Rates of quinone reduction were obtained by monitoring the absorbance at 340 nm for oxidation of either NADPH (paAzoR1) or NADH (paAzoR2 and paAzoR3). |
| GO:0009055 electron transfer activity | IEA GO_REF:0000104 | MARK AS OVER ANNOTATED | Summary: UniRule mapping. paAzoR2 is an FMN enzyme, not an electron carrier. Reason: Electron transfer through the bound FMN is part of the catalytic activity, not an electron-carrier function. Same call as for azoR1 and PA0949. Supporting Evidence: PMID:24915188 Rates of quinone reduction were obtained by monitoring the absorbance at 340 nm for oxidation of either NADPH (paAzoR1) or NADH (paAzoR2 and paAzoR3). |
| GO:0010181 FMN binding | IEA GO_REF:0000120 | ACCEPT | Summary: FMN cofactor of the azoreductase type 1 family (HAMAP MF_01216), shown structurally for the paralog paAzoR1 and modelled for paAzoR2. Reason: FMN is the redox cofactor of every characterised member of the family, and Ryan et al. modelled the FMN-binding cradle in paAzoR2. Supporting Evidence: PMID:20417637 We have defined a novel flavin mononucleotide binding cradle, which is a recurrent motif in many flavodoxin-like proteins. |
| GO:0016652 oxidoreductase activity, acting on NAD(P)H as acceptor | IEA GO_REF:0000104 | REMOVE | Summary: UniRule mapping to a term whose definition has NAD(P)+ as the electron acceptor (EC 1.6.1). paAzoR2 uses NADH as donor and quinones or azo compounds as acceptors. Reason: The mapping is demonstrably wrong for azoreductases. The UNIPROT source defect for UniRule UR000101231 is recorded once, on the azoR1 review. Supporting Evidence: PMID:24915188 Rates of quinone reduction were obtained by monitoring the absorbance at 340 nm for oxidation of either NADPH (paAzoR1) or NADH (paAzoR2 and paAzoR3). |
| GO:0016655 oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor | IBA GO_REF:0000033 | TOO GENERAL | Summary: IBA from PTN000024600, seeded by E. coli AzoR (EcoCyc IDA PMID:19666717) and by the three P. aeruginosa azoreductases (PseudoCAP IDA PMID:24915188). azoR2 appears in its own WITH/FROM, as expected. Reason: Correct inheritance; the IBD stands on E. coli AzoR alone. GO:0003955, asserted as a NEW row here, is_a this term. Supporting Evidence: PMID:19666717 It was found that AzoR was capable of reducing several benzo-, naphtho-, and anthraquinone compounds, which were better substrates for AzoR than the model azo substrate methyl red. |
| GO:0016655 oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor | IDA PMID:24915188 Identification of NAD(P)H quinone oxidoreductase activity in... | TOO GENERAL | Summary: PseudoCAP IDA and one of four seeds of the PTN000024600 IBD. Ryan et al. purified paAzoR2 and measured NADH-dependent reduction of a quinone panel. Seed verdict SOUND. Reason: The paper (full text read) directly supports quinone reductase activity of the purified enzyme. GO:0003955, asserted as a NEW row from the same paper, is the specific child. Supporting Evidence: PMID:24915188 These maximum rates were as follows: paAzoR1 and paAzoR2 reducing Bzq 0.57 mM.s−1.mg of enzyme−1 and 7.78 mM.s−1.mg of enzyme−1 respectively and paAzoR3 reducing Plu 16.8 mM.s−1.mg of enzyme−1. |
| GO:0016655 oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro2GO from the FMN-dependent NADH:quinone oxidoreductase signature. Reason: Correct; GO:0003955, asserted as a NEW row in this review, is the specific child. Supporting Evidence: PMID:24915188 Rates of quinone reduction in all cases were up to two orders of magnitude higher than were observed for reduction of azo substrates |
| GO:0003955 NAD(P)H dehydrogenase (quinone) activity | IDA PMID:24915188 Identification of NAD(P)H quinone oxidoreductase activity in... | NEW | Summary: Proposed specific term for the measured activity: purified paAzoR2 oxidises NADH while reducing benzoquinones and naphthoquinones, and the paper classes the azoreductases with the two-electron NAD(P)H quinone oxidoreductases. Reason: Direct enzyme assay on the purified protein; the core molecular function. Same call as for azoR1 and azoR3. Supporting Evidence: PMID:24915188 paAzoR2 reduces both benzoquinones and naphthoquinones efficiently PMID:24915188 Two electron reduction reactions of quinones are carried out by a group of enzymes known as NAD(P)H quinone oxidoreductases. |
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Download this section (compressed HTML)Q: azoR2, unlike azoR1 and azoR3, lacks the response to oxidative stress IBA from PTN000811013. Does it sit outside that subnode, and does it have a distinct physiological substrate?
Experiment: Compare benzoquinone and naphthoquinone sensitivity of azoR2 single and azoR1 azoR2 azoR3 triple deletion mutants of PAO1.
Type: genetic / stress survival
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