azoR1 (PA0785) encodes paAzoR1, a soluble FMN-dependent flavodoxin-fold NAD(P)H:quinone oxidoreductase of the azoreductase type 1 family. The purified enzyme binds one FMN per subunit, forms dimers that associate into tetramers, and uses NADPH (and NADH) to reduce quinones, with a preference for benzoquinones, at rates far above those for azo compounds. It also reduces azo dyes and azo pro-drugs (balsalazide, sulfasalazine, olsalazine) and the nitrofuran antibiotic nitrofurazone. Crystal structures with methyl red, balsalazide, nitrofurazone and quinones show a hydrophobic active site formed between two monomers above the FMN. By analogy with Escherichia coli AzoR, its physiological role is thought to be two-electron reduction of electrophilic quinones to quinols, 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:0003960 quinone reductase (NADPH) activity | EXP PMID:24915188 Identification of NAD(P)H quinone oxidoreductase activity in... | MODIFY | Summary: UniProt EXP row from Ryan et al. 2014, which measured NADPH oxidation by purified paAzoR1 in the presence of quinones. The quinone reductase claim is right; the chosen GO term defines a one-electron reaction that yields semiquinones. Reason: GO:0003960 is defined as 2 quinone + NADPH = 2 1,4-benzosemiquinone + NADP+ (RHEA:14269), the one-electron reaction of zeta-crystallin-type enzymes. paAzoR1 is an FMN flavoenzyme that transfers a hydride, and the paper places it among the two-electron NAD(P)H quinone oxidoreductases that make quinols. The paper did not identify the product, so this is a lateral correction to GO:0003955, which states the quinol-forming reaction, rather than a claim that semiquinone formation was disproved. Proposed replacements: NAD(P)H dehydrogenase (quinone) activity Supporting Evidence: PMID:24915188 Two electron reduction reactions of quinones are carried out by a group of enzymes known as NAD(P)H quinone oxidoreductases. 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:0003960 quinone reductase (NADPH) activity | IEA GO_REF:0000116 | MODIFY | Summary: Rhea2GO mapping from the UniProt CATALYTIC ACTIVITY line (RHEA:14269), the same claim as the EXP row. Reason: As for the EXP row, the quinone reductase activity is real but the semiquinone-forming reaction is a poor fit for an FMN hydride-transfer enzyme; GO:0003955 is the quinol-forming term. Proposed replacements: NAD(P)H dehydrogenase (quinone) activity Supporting Evidence: PMID:24915188 Two electron reduction reactions of quinones are carried out by a group of enzymes known as NAD(P)H quinone oxidoreductases. |
| GO:0006979 response to oxidative stress | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA from PTN000811013, seeded only by E. coli AzoR (EcoCyc IMP, PMID:11583992). E. coli AzoR mutants lose viability under electrophilic quinone stress. Reason: A plausible inherited role: quinone reductases that make quinols prevent redox cycling, and the E. coli ortholog protects against electrophilic quinones. The E. coli phenotype is thiol-specific (electrophile) stress rather than reactive oxygen stress, and no P. aeruginosa mutant phenotype is known, so this is kept as a non-core inference. Supporting Evidence: PMID:19666717 These data indicated that AzoR is a quinone reductase providing resistance to thiol-specific stress caused by electrophilic quinones. PMID:24915188 The quinones identified in this study appear to be the best substrates for the enzymes yet identified, indicating that they may act as the physiological substrate for these enzymes. |
| GO:0009055 electron transfer activity | IEA GO_REF:0000104 | MARK AS OVER ANNOTATED | Summary: UniRule mapping. paAzoR1 is an enzyme that reduces its substrates with a bound FMN; it is not an electron carrier that shuttles electrons between redox partners. Reason: The FMN mediates electron transfer within the enzyme, which is part of the catalytic activity, not an electron-carrier function. Same call as for the flavodoxin-fold quinone reductase PA0949. Supporting Evidence: PMID:17904577 During azo reduction, FMN serves as a redox centre in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate. |
| GO:0010181 FMN binding | IEA GO_REF:0000120 | ACCEPT | Summary: FMN cofactor binding, shown biochemically and in several crystal structures of paAzoR1. Reason: The purified protein carries one non-covalently bound FMN per subunit. Supporting Evidence: PMID:17904577 Each recombinant protein molecule has an estimated molecular mass of 23,050 Da and one non-covalently bound FMN as co-factor. |
| 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 (transhydrogenase-type, EC 1.6.1). AzoR enzymes use NAD(P)H as the donor and quinones, azo or nitro compounds as acceptors. Reason: GO:0016652 is defined as NADH or NADPH reducing NAD+ or NADP. No azoreductase reduces a pyridine nucleotide. The mapping probably comes from the EC 1.7.1.17 reaction, which is written in the reverse direction with NAD+ as acceptor; EC 1.7.1 is not EC 1.6.1. The mapping is demonstrably wrong. Supporting Evidence: PMID:17904577 During azo reduction, FMN serves as a redox centre in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate. |
| 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). azoR1 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 from the same paper, is_a this term and names the quinol-forming reaction. 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 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 paAzoR1 and measured NADPH-dependent reduction of a panel of benzoquinones and naphthoquinones. 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 The pure recombinant proteins paAzoR1 (PA0785), paAzoR2 (PA1962) and paAzoR3 (PA3223) were expressed and purified as described previously [15], [21]. 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 paAzoR1 oxidises NADPH 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, with quinone-bound crystal structures, makes this the core molecular function. It replaces the general GO:0016655 rows and the semiquinone-forming GO:0003960 rows. 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). 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: Is the product of quinone reduction by paAzoR1 the quinol (two-electron) or the semiquinone, as the UniProt Rhea reaction states?
Q: GO has no term for FMN-dependent NADH-azoreductase activity (EC 1.7.1.17); only GO:0050446 azobenzene reductase (NADP+) exists. Should one be requested for the azoreductase type 1 family?
Experiment: Delete azoR1, azoR2 and azoR3 singly and together in PAO1 and test survival under benzoquinone, menadione, plumbagin and homogentisate challenge, to see whether the measured quinone reductase activity protects the cell as E. coli AzoR does.
Type: genetic interaction / stress survival
Experiment: Identify the reduction product (quinol versus semiquinone) by anaerobic spectroscopy or EPR during steady-state turnover with benzoquinone.
Type: enzyme mechanism
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