PhzS is the 5-methylphenazine-1-carboxylate 1-monooxygenase (EC 1.14.13.218) of Pseudomonas aeruginosa PAO1, an FAD-dependent class A aromatic hydroxylase of the p-hydroxybenzoate hydroxylase family. It uses NADH and O2 to catalyse the hydroxylative decarboxylation of 5-methylphenazine-1-carboxylate, the product of the PhzM N-methyltransferase, giving the blue redox-active virulence pigment pyocyanin. This is the last step in converting phenazine-1-carboxylate (PCA) to pyocyanin. PhzS can also hydroxylate PCA itself to 1-hydroxyphenazine, but PCA is a poor substrate that largely uncouples NADH oxidation from hydroxylation. The purified enzyme is a monomer with bound FAD; its crystal structure shows the flavin in the "out" conformation and a disordered C-terminal tail near the substrate tunnel. In vitro, pyocyanin is formed only when PhzM and PhzS are free to meet, which suggests a transient PhzM-PhzS interaction that keeps the unstable 5-methyl-PCA intermediate from being released. phzS insertion mutants make no pyocyanin.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004497 monooxygenase activity | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic inference from PTN002492679, the LUCA root of the PTHR13789 FAD-dependent monooxygenase family (104 reference-genome leaves, mostly fungal salicylate-hydroxylase-like proteins plus bacterial and algal members). Its only seed is PhzS itself. The claim is true for PhzS: purified PhzS hydroxylates PCA NADH-dependently and makes pyocyanin with PhzM. For this gene the term is correct but general, because the specific reaction is known and already annotated (GO:0102169, an is_a descendant of GO:0004497 via GO:0016709). Reason: Correct but less specific than the evidence supports. The node-level generic term is the right level for the clade, but on PhzS the supported specific activity is GO:0102169, which the gene already carries by EXP. PhzS appearing in its own with/from is expected (it is the seed). Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002492679 · PTN002492679 SUPPORTS TRANSFER Family-root IBD of the generic class A flavoprotein monooxygenase activity; appropriate breadth for the clade. One leaf (Synechocystis chlP, Q55087, a geranylgeranyl reductase) is a likely exception. UniProtKB:Q9HWG9 · phzS (PA4217) SUPPORTS TRANSFER Seed. Monooxygenase activity is shown in PMID:17253782 (and the UniProt EXP GO:0102169 row), although the PseudoCAP seed row cites a crystallization paper with no activity assay. Proposed replacements: 5-methylphenazine-1-carboxylate 1-monooxygenase (NADH) activity Supporting Evidence: PMID:17253782 This was confirmed by incubating PhzS with PCA and NADH. 1-HP was formed in an NADH dependant manner PMID:18416536 The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. file:PSEAE/phzS/phzS-bioinformatics/RESULTS.md PTN002492679 is the root of the PTHR13789 tree (LUCA speciation node, is_family_root: True), 104 reference-genome leaves in 19 organisms. |
| GO:0004497 monooxygenase activity | IDA PMID:17012792 The purification, crystallization and preliminary structural... | MODIFY | Summary: PseudoCAP IDA citing Gohain et al. 2006, a crystallization communication (full text cached). The paper purifies His-tagged PhzS, crystallizes it and collects diffraction data; it calls PhzS an FAD-dependent monooxygenase from earlier genetics and fold recognition, and notes the yellow FAD colour of the crystals, but it measures no enzyme activity. The claim is nevertheless true (PMID:17253782 shows NADH-dependent hydroxylation by purified PhzS), and it is general relative to the known reaction. This row is the sole seed of the PTN002492679 IBD. Reason: The function is correct but general; the specific supported activity is GO:0102169, already present by EXP. The cited paper does not assay activity, so the row should be re-keyed to PMID:17253782 (recorded as a PSEUDOCAP source defect). Kept consistent with the IBA row for the same term. Proposed replacements: 5-methylphenazine-1-carboxylate 1-monooxygenase (NADH) activity Supporting Evidence: PMID:17253782 Pyocyanin formation required PhzM, PhzS, PCA, SAM, oxygen, and NADH consistent with a methyl transfer reaction and a flavin-mediated hydroxylation. PMID:17012792 The crystals are yellow owing to the presence of FAD |
| GO:0005515 protein binding | IPI PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | REMOVE | Summary: Interaction with PhzM inferred from activity assays: pyocyanin forms only when PhzM and PhzS can meet, not when separated by a dialysis membrane or sequentially by filtration. No stable PhzM-PhzS complex was detected by light scattering. The bare protein-binding term says nothing about the function. Reason: Generic protein binding is uninformative (protein-binding policy). Removal does not deny the reported functional PhzM-PhzS interaction, which is recorded in the description and core function. No more informative MF term fits a transient, inferred encounter. Supporting Evidence: PMID:17253782 Isolation of either PhzM or PhzS inside a dialysis membrane results in no pyocyanin formation suggesting that an at least transient physical interaction between PhzM and PhzS is required in order to activate production of pyocyanin. PMID:17253782 Elution profiles and calculated masses did not differ from the data obtained with buffer only in the system indicating detectable complexes were not formed under these conditions. |
| GO:0016709 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning prediction of an NAD(P)H-dependent monooxygenase-type oxidoreductase. Correct for PhzS (NADH and O2 are required), but GO:0102169, an is_a child of this term, is the supported specific activity. Reason: True but general; replace with the specific descendant GO:0102169 that the gene already carries by EXP. Proposed replacements: 5-methylphenazine-1-carboxylate 1-monooxygenase (NADH) activity Supporting Evidence: file:PSEAE/phzS/phzS-uniprot.txt Catalyzes the oxidative decarboxylation of 5-methylphenazine-1-carboxylate (5-methyl- PCA) to pyocyanine. |
| GO:0071949 FAD binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro FAD-binding domain mapping. Correct: the purified enzyme and both crystal structures contain bound FAD, and the FAD-binding residues are defined experimentally. Reason: Cofactor binding that is as precise as warranted and is part of, but peripheral to, the core monooxygenase activity. Supporting Evidence: file:PSEAE/phzS/phzS-uniprot.txt Name=FAD; Xref=ChEBI:CHEBI:57692; Evidence={ECO:0000269|PubMed:18416536, ECO:0000269|Ref.5}; PMID:18416536 The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. |
| GO:0102169 5-methylphenazine-1-carboxylate 1-monooxygenase (NADH) activity | EXP PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | ACCEPT | Summary: Purified PhzM plus PhzS, with PCA, SAM, NADH and O2, produce pyocyanin; PhzM alone is inactive and PhzS alone converts PCA only to 1-hydroxyphenazine. The final step (5-methyl-PCA to pyocyanin) is assigned to the flavin hydroxylase PhzS. The free intermediate could not be assayed directly because it is unstable, so the reaction is shown in the coupled system. Reason: Core catalytic activity of PhzS, supported by the coupled in vitro reconstitution and by curated UniProt function and catalytic activity statements tied to this paper. Supporting Evidence: PMID:17253782 When reactions containing both PhzM and PhzS were analyzed, rapid pyocyanin formation was evident based on the blue color formed in the reaction vessel. PMID:17253782 Pyocyanin formation required PhzM, PhzS, PCA, SAM, oxygen, and NADH consistent with a methyl transfer reaction and a flavin-mediated hydroxylation. file:PSEAE/phzS/phzS-uniprot.txt Catalyzes the oxidative decarboxylation of 5-methylphenazine-1-carboxylate (5-methyl- PCA) to pyocyanine. |
| GO:0102169 5-methylphenazine-1-carboxylate 1-monooxygenase (NADH) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Rhea/EC-based electronic annotation for EC 1.14.13.218, which agrees with the experimental row for the same term. Reason: Consistent with the experimentally supported core activity. Supporting Evidence: file:PSEAE/phzS/phzS-uniprot.txt Reaction=5-methyl-phenazine-1-carboxylate + NADH + O2 + 2 H(+) = pyocyanin + CO2 + NAD(+) + H2O |
| GO:0106220 pyocyanine biosynthetic process | IDA PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | NEW | Summary: Not in GOA for PhzS, though its partner PhzM carries this term by IDA from the same paper. PhzS itself catalyses the final step of pyocyanin synthesis, and phzS insertion mutants lack pyocyanin. Reason: PhzS does the work of a step in this process: it is the enzyme that converts 5-methyl-PCA to pyocyanin. This is participation, not only necessity. Comparator: PhzM, the enzyme of the preceding step, carries GO:0106220 by IDA from PMID:17253782, and UniProt's curated pathway line places PhzS in pyocyanine biosynthesis (ECO:0000269). Supporting Evidence: PMID:17253782 Pyocyanin formation required PhzM, PhzS, PCA, SAM, oxygen, and NADH consistent with a methyl transfer reaction and a flavin-mediated hydroxylation. PMID:11591691 P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. file:PSEAE/phzS/phzS-uniprot.txt PATHWAY: Secondary metabolite biosynthesis; pyocyanine biosynthesis. |
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Download this section (compressed HTML)Q: Is the PhzM-PhzS interaction a defined complex that channels the unstable 5-methyl-PCA intermediate, or a transient encounter? Which PhzS surface (for example the disordered C-terminal tail) mediates it?
Q: Should PseudoCAP re-key the PhzS monooxygenase IDA row from the crystallization paper PMID:17012792 to PMID:17253782? And should Synechocystis chlP (Q55087, a geranylgeranyl reductase) be removed from PTHR13789, or given NOT monooxygenase activity, so that it no longer inherits the PTN002492679 IBA?
Experiment: Make 5-methyl-PCA chemically or trap it enzymatically, and measure PhzS steady-state kinetics on it directly, with and without PhzM. This would show whether PhzM changes PhzS activity or only supplies the substrate.
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