ubiH (PP_5199) encodes a class A flavoprotein monooxygenase of the UbiH/COQ6 family that uses FAD, NAD(P)H and O2 to hydroxylate the aromatic ring of a ubiquinone intermediate. By orthology to Escherichia coli UbiH it introduces the C-1 (para) hydroxyl of 2-polyprenyl-6-methoxyphenol, giving 2-polyprenyl-6-methoxy-1,4-benzoquinol, the substrate of the UbiE C-methyltransferase. It is one of the three oxygen-dependent ring hydroxylases of Pseudomonas putida, together with the UbiI-type VisC (PP_5197, encoded two genes away) and the di-iron Coq7 (PP_0427), which replaces the UbiF hydroxylase of E. coli. The assignment rests on UbiH-specific family signatures and sequence similarity; the KT2440 protein has not been assayed.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000117 | MODIFY | Summary: ARBA root-level MF. True but uninformative; the specific hydroxylase activity is carried by this gene. Reason: Catalytic activity is a correct root ancestor of the UbiH hydroxylase activity (GO:0008681), which this gene already carries from the InterPro/PANTHER pipeline and which is supported by UbiH-specific signatures. Refine to that term. Proposed replacements: 2-octaprenyl-6-methoxyphenol hydroxylase activity Supporting Evidence: file:PSEPK/ubiH/ubiH-uniprot.txt DR NCBIfam; TIGR01984; UbiH; 1. file:PSEPK/visC/visC-bioinformatics/RESULTS.md Q88CI2 (PP_5199) is most similar to E. coli UbiH, consistent with its UbiH-specific |
| GO:0006744 ubiquinone biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Core process. UbiH catalyses one of the three ring hydroxylations of ubiquinone biosynthesis. Reason: UbiH performs a pathway step. In E. coli UbiH is the C-1 hydroxylase of the pathway, and Pseudomonas aeruginosa, a close relative, uses the UbiH + UbiI + Coq7 hydroxylase set. Q88CI2 is the UbiH member of that set in KT2440 (UbiH-specific NCBIfam TIGR01984 and InterPro IPR011295; closest E. coli homolog UbiH). PAINT places GO:0006744 at PTHR43876 node PTN008453628, the ancestor of the bacterial UbiH, UbiI and UbiF clades. Supporting Evidence: PMID:27822549 Prior to our study, four UQ hydroxylases (the di-iron monooxygenase Coq7 and the three FMOs UbiF, UbiH, and UbiI) had been characterized, constituting two different protein sets in E. coli (UbiF, UbiH, and UbiI) (11) and in P. aeruginosa and A. ferrooxidans (UbiH, UbiI, and Coq7) (16). file:PSEPK/ubiH/ubiH-uniprot.txt DR InterPro; IPR011295; UbiH. file:PSEPK/ubiH/ubiH-deep-research-openscientist.md The gene is annotated as *ubiH* in UniProt, KEGG (ppu:PP_5199, orthology K03185, EC 1.14.13.-), and InterPro |
| GO:0008681 2-octaprenyl-6-methoxyphenol hydroxylase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Core molecular function, from InterPro IPR011295 (UbiH) and the PANTHER UbiH node (PAINT IBD at PTN000350242, seed E. coli UbiH P25534). Accepted with a chain-length caveat. Reason: This is the UbiH reaction (C-1 hydroxylation of 2-polyprenyl-6-methoxyphenol). Q88CI2 carries the UbiH-specific NCBIfam TIGR01984 and the PANTHER subfamily PTHR43876:SF8, is closest to E. coli UbiH in pairwise identity (40.5% vs 34-36% to UbiI/UbiF), and the regioselectivity of UbiH is expected to be conserved across beta- and gammaproteobacteria. The GO term as defined names the octaprenyl substrate of E. coli; KT2440 makes ubiquinone-9, so its physiological substrate is the nonaprenyl homolog. GO has no chain-length-neutral NAD(P)H-dependent term for this step (GO:0120538 is the ferredoxin-dependent version), so GO:0008681 is the closest available term. Supporting Evidence: PMID:27822549 (iii) Both Coq7 and UbiF also co-occur with UbiH and UbiI, which in E. coli have preferences for C-1 and C-5, respectively (11, 20), and we expect the regioselectivities of UbiH and UbiI to be conserved in most beta- and gammaproteobacterial proteins, given their strong co-occurrence properties (Fig. S6). file:PSEPK/ubiH/ubiH-uniprot.txt DR PANTHER; PTHR43876:SF8; 2-OCTAPRENYL-6-METHOXYPHENOL HYDROXYLASE; 1. file:PSEPK/visC/visC-bioinformatics/RESULTS.md Q88CI2 (PP_5199) is most similar to E. coli UbiH, consistent with its UbiH-specific |
| GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO family-level term from IPR010971 (UbiH/COQ6). True but general; refine to the specific UbiH activity carried by this gene. Reason: Class A flavoprotein monooxygenases use NAD(P)H and O2, so the paired-donor oxygenase term is correct, but the family covers UbiH, UbiI, UbiF and Coq6 with different regioselectivities. For a UbiH-clade member the specific term GO:0008681 is supported and already carried. Proposed replacements: 2-octaprenyl-6-methoxyphenol hydroxylase activity Supporting Evidence: PMID:27822549 Class A FMOs are a widely distributed subset of FMOs that use a flavin adenine dinucleotide (FAD) cofactor, NAD(P)H as the electron donor, and dioxygen in order to catalyze aromatic hydroxylation reactions (12). |
| GO:0050660 flavin adenine dinucleotide binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Cofactor binding inferred from the UbiH/COQ6 family. True (class A FMOs carry FAD and cycle it between oxidized and reduced forms), but a cofactor property, not the core activity. Reason: Q88CI2 has the FAD_binding_3 (PF01494) domain of class A flavoprotein monooxygenases, which bind FAD and reduce it with NAD(P)H during catalysis; GO:0050660 covers both oxidized and reduced FAD. It is as precise as warranted for a cofactor-binding statement and is peripheral to the hydroxylase function. Supporting Evidence: file:PSEPK/ubiH/ubiH-uniprot.txt DR Pfam; PF01494; FAD_binding_3; 1. PMID:27822549 Class A FMOs are a widely distributed subset of FMOs that use a flavin adenine dinucleotide (FAD) cofactor, NAD(P)H as the electron donor, and dioxygen in order to catalyze aromatic hydroxylation reactions (12). |
| GO:0071949 FAD binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO from the FAD-binding domain IPR002938. True and non-core, consistent with the GO:0050660 row. Reason: The resting enzyme of PHBH-fold monooxygenases binds oxidized FAD non-covalently; the E. coli paralog UbiI was crystallized with this fold and needs flavin-pocket residues for activity. Cofactor binding is non-core relative to the hydroxylase activity. Supporting Evidence: file:PSEPK/ubiH/ubiH-uniprot.txt DR InterPro; IPR002938; FAD-bd. PMID:23709220 This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. |
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Download this section (compressed HTML)Q: GO:0008681 is defined on the octaprenyl substrate of E. coli. Should GO add a chain-length-neutral NAD(P)H-dependent 2-polyprenyl-6-methoxyphenol 4-hydroxylase term (parallel to GO:0019168 and GO:0008682) for UbiH proteins of organisms making UQ-9 or UQ-10?
Q: Does PP_5199 retain strict C-1 regioselectivity, or can it, like the generalist UbiL/UbiM enzymes, hydroxylate a second ring position?
Experiment: Express PP_5199 in an E. coli ubiH deletion strain and test restoration of UQ-8 and aerobic growth on succinate; delete PP_5199 in KT2440 and profile accumulated 2-nonaprenyl-6-methoxyphenol.
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