PhzM is the phenazine-1-carboxylate N-methyltransferase (EC 2.1.1.327) of Pseudomonas aeruginosa PAO1. It transfers a methyl group from S-adenosyl-L-methionine to N5 of phenazine-1-carboxylate (PCA), giving 5-methylphenazine-1-carboxylate. The flavin monooxygenase PhzS then converts this unstable intermediate into the blue redox-active virulence pigment pyocyanin. PhzM is a class I SAM-dependent methyltransferase related to plant O-methyltransferases such as isoflavone O-methyltransferase. It is a homodimer: an N-terminal dimerization domain forms an intertwined interface that also lines the partner subunit's active site, and a C-terminal Rossmann domain carries the SAM-binding motif. Purified PhzM alone shows no detectable activity on PCA. Pyocyanin is made readily when PhzS and NADH are present, and only when the two enzymes can meet, which suggests that a transient PhzM-PhzS interaction activates methylation and keeps the intermediate from being released. phzM insertion mutants lack pyocyanin, and phzM expression is a control point for pyocyanin output (for example by Crc binding to phzM mRNA).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | REMOVE | Summary: Interaction with PhzS inferred from activity: no pyocyanin forms when PhzM and PhzS are separated by a dialysis membrane or removed by filtration before the partner is added. Light scattering found no stable complex. Bare protein binding carries no functional information. Reason: Generic protein binding is uninformative (protein-binding policy). Removal does not deny the functional PhzM-PhzS interaction, which is described in the description and core function. 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. |
| GO:0008168 methyltransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro COMT-like family mapping. Correct but general; the specific reaction (PCA N5-methylation) is known and annotated. Reason: True but general; GO:0102168, an is_a child of GO:0008168, is the supported specific activity and is already present by IDA. Proposed replacements: 5-methyl-phenazine-1-carboxylate N-methyltransferase activity Supporting Evidence: file:PSEAE/phzM/phzM-uniprot.txt Converts phenazine-1-carboxylate (PCA) to 5-methylphenazine-1-carboxylate (5- methyl-PCA). |
| GO:0008171 O-methyltransferase activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping from the O-methyltransferase C-terminal domain (COMT_C). The family is named for its many O-methylating members, but PhzM methylates a ring nitrogen (N5) of PCA, not an oxygen. No O-methyltransferase activity has been reported for PhzM. Reason: Demonstrably wrong electronic mapping for this member: a family-level O-methyltransferase term applied to an N-methyltransferase. The structural paper contrasts PhzM's N5 methylation with the O-methylation of its plant homologue. Supporting Evidence: PMID:17253782 isoformononetin (also a tricyclic compound) is methylated on an oxygen at the end of its long axis while PCA is methylated at N5 in the center of the molecule file:PSEAE/phzM/phzM-uniprot.txt Reaction=phenazine-1-carboxylate + S-adenosyl-L-methionine |
| GO:0008757 S-adenosylmethionine-dependent methyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference from PTN002463933, the LUCA root of PTHR43712 (80 reference-genome leaves; mostly fungal and animal O-methyltransferases plus a small bacterial PhzM-like clade). PhzM is the only seed. The claim is directly supported for PhzM. In current GO, the specific reaction term GO:0102168 is not a descendant of GO:0008757, so this term adds the SAM dependence that the specific term does not entail. Reason: Core activity; the node term is appropriately general for a clade that mixes O- and N-methyltransferases. PhzM in its own with/from is expected (it is the seed). Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002463933 · PTN002463933 SUPPORTS TRANSFER Family-root IBD of SAM-dependent methyltransferase activity; right breadth for the clade. Two fungal cluster coactivators (tpcD, mdpA) inherit it and may be non-catalytic; not checked. UniProtKB:Q9HWH2 · phzM (PA4209) SUPPORTS TRANSFER Seed; SAM-dependent methyl transfer to PCA shown in the coupled PhzM-PhzS assay (PMID:17253782). 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. file:PSEAE/phzM/phzM-bioinformatics/RESULTS.md PTN002463933 is the root of the PTHR43712 tree (LUCA speciation node, is_family_root: True), 80 reference-genome leaves in 16 organisms. |
| GO:0008757 S-adenosylmethionine-dependent methyltransferase activity | IDA PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | ACCEPT | Summary: PseudoCAP IDA, the sole seed of the PTN002463933 IBD. Parsons et al. reconstituted pyocyanin synthesis from PCA with purified PhzM and PhzS: product formation needed SAM as well as both enzymes, NADH and O2. PhzM alone did not consume SAM or PCA, which the authors attribute to a need for activation by PhzS rather than to a lack of activity. The class I methyltransferase fold and the DVGGGXG SAM motif assign the methyl transfer to PhzM, not to the flavin hydroxylase PhzS. Reason: The paper directly supports SAM-dependent methyl transfer by PhzM in a defined system with purified proteins; this is the gene's own catalytic activity, not a mutant phenotype. The seed is sound. 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:17253782 PhzM possesses the highly conserved DVGGGXG motif (residues 173–179) that defines the SAM binding site of methyltransferase enzymes suggesting that PhzM does indeed function as a methyltransferase. PMID:17253782 Incubations of PhzM with its presumed substrates, PCA, and SAM, yielded no detectable product and substrate concentrations were unchanged after extended incubations. |
| GO:0042802 identical protein binding | IDA PMID:16946471 The purification, crystallization and preliminary structural... | MODIFY | Summary: PhzM self-association from the crystallization study (one dimer per asymmetric unit; described as dimeric). True, but less informative than the homodimerization term that the same papers also support. Reason: Correct but general; GO:0042803 protein homodimerization activity, an is_a child already present by IDA, is the supported specific term. Proposed replacements: protein homodimerization activity Supporting Evidence: PMID:16946471 PhzM, a dimeric S-adenosylmethionine-dependent methyltransferase, was purified and crystallized |
| GO:0042802 identical protein binding | IDA PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | MODIFY | Summary: Light scattering shows PhzM is a dimer in solution, and the crystal structure shows an extensive intertwined dimer interface. True but less specific than homodimerization. Reason: Correct but general; replace with GO:0042803, already present by IDA. Proposed replacements: protein homodimerization activity Supporting Evidence: PMID:17253782 Laser light scattering confirms that PhzM is a dimer in solution |
| GO:0042803 protein homodimerization activity | IDA PMID:16946471 The purification, crystallization and preliminary structural... | KEEP AS NON CORE | Summary: Crystals contain one PhzM dimer per asymmetric unit, and the protein is described as dimeric (abstract only in cache; consistent with the later solution data). Reason: Supported structural property. The dimer interface contributes to the partner subunit's active site, but dimerization is a feature of the enzyme rather than its core function. Supporting Evidence: PMID:16946471 They contain one dimer in the asymmetric unit |
| GO:0042803 protein homodimerization activity | IDA PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | KEEP AS NON CORE | Summary: Dimer in solution by laser light scattering (about twice the monomer mass), with a domain I-mediated intertwined interface in the structure. Reason: Directly supported, but peripheral to the methyltransferase function. Supporting Evidence: PMID:17253782 Laser light scattering confirms that PhzM is a dimer in solution as well with a molecular mass of 73550 |
| GO:0046983 protein dimerization activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro COMT-like dimerisation domain mapping. Correct, but the experiments show the specific case, homodimerization. Reason: True but general; GO:0042803 (is_a descendant) is supported by IDA in two papers. Proposed replacements: protein homodimerization activity Supporting Evidence: PMID:17253782 Laser light scattering confirms that PhzM is a dimer in solution |
| GO:0102168 5-methyl-phenazine-1-carboxylate N-methyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Rhea/EC-based electronic annotation for EC 2.1.1.327. Agrees with the IDA row for the same term. Reason: Consistent with the experimentally supported core activity. Supporting Evidence: file:PSEAE/phzM/phzM-uniprot.txt Reaction=phenazine-1-carboxylate + S-adenosyl-L-methionine |
| GO:0102168 5-methyl-phenazine-1-carboxylate N-methyltransferase activity | IDA PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | ACCEPT | Summary: The PCA N5-methylation reaction. It was shown in the coupled system (PhzM + PhzS + SAM + NADH + O2 giving pyocyanin), because the free 5-methyl-PCA product is unstable and PhzM alone is inactive. Curated UniProt catalytic activity cites this paper. Reason: Core catalytic activity, the most specific supported MF term for PhzM. 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. file:PSEAE/phzM/phzM-uniprot.txt Converts phenazine-1-carboxylate (PCA) to 5-methylphenazine-1-carboxylate (5- methyl-PCA). |
| GO:0106220 pyocyanine biosynthetic process | IDA PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | ACCEPT | Summary: PhzM catalyses the first of the two steps from PCA to pyocyanin, and in vitro reconstitution with PhzS produces pyocyanin. phzM mutants lack pyocyanin. Reason: Core process in which PhzM catalyses a step. 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:11591691 P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. file:PSEAE/phzM/phzM-uniprot.txt PATHWAY: Secondary metabolite biosynthesis; pyocyanine biosynthesis. |
| GO:1904047 S-adenosyl-L-methionine binding | IDA PMID:17253782 Structural and functional analysis of the pyocyanin biosynth... | KEEP AS NON CORE | Summary: True, since SAM is the methyl donor and PhzM carries the DVGGGXG SAM-binding motif. But the structure in this paper is unliganded, and no binding assay is reported: SAM binding is inferred from the SAM requirement of the coupled reaction and from the motif. Reason: Cosubstrate binding is part of the core methyltransferase activity rather than a function in its own right. Kept, but the direct-assay evidence for binding is weak. Supporting Evidence: PMID:17253782 The structure of PhzM contains no ligands and the active site is open and solvent exposed when compared to structures of similar enzymes. PMID:17253782 PhzM possesses the highly conserved DVGGGXG motif (residues 173–179) that defines the SAM binding site of methyltransferase enzymes |
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Download this section (compressed HTML)Q: Should GO:0102168 (5-methyl-phenazine-1-carboxylate N-methyltransferase activity), currently only is_a GO:0008168, be placed under GO:0008757 S-adenosylmethionine-dependent methyltransferase activity and GO:0008170 N-methyltransferase activity, as its SAM-dependent reaction requires?
Q: How does PhzS activate PhzM, and is 5-methyl-PCA channelled between them?
Q: Do the PTHR43712 cluster coactivators tpcD (Q4WQZ4) and mdpA (C8VQ72) keep the SAM-binding and catalytic residues? If not, they should not inherit the PTN002463933 methyltransferase IBA.
Experiment: Co-crystallize PhzM with SAH and PCA, or a PhzM-PhzS complex trapped by cross-linking, and test PhzM activity with catalytically inactive PhzS variants. This would separate activation by binding from activation by product removal.
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