PhnB is the small, glutamine amidotransferase subunit (component II) of the second anthranilate synthase of Pseudomonas aeruginosa PAO1, encoded in the phnAB operon. It is a 200-residue protein consisting of a single class I (triad) glutamine amidotransferase domain with a Cys-His-Glu catalytic triad; it hydrolyses glutamine and channels the resulting ammonia to the large subunit PhnA, which condenses it with chorismate to give anthranilate. The two subunits form a heterotetramer. PAO1 carries a second, independently regulated anthranilate synthase, TrpEG, which serves tryptophan biosynthesis; the two enzymes are biochemically interchangeable but differ in regulation, PhnAB being expressed at higher cell density and not feedback-inhibited by tryptophan. The anthranilate PhnAB makes is the precursor of the 4-quinolone signal PQS (2-heptyl-3-hydroxy-4-quinolone) and related alkyl quinolones; because PQS induces the phenazine operons, loss of phnAB lowers pyocyanin output indirectly. The phenazine ring itself is built from chorismate by PhzE through 2-amino-2-deoxyisochorismate and does not pass through anthranilate.
Definition: The chemical reactions and pathways resulting in the formation of 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal, PQS), an alkyl quinolone signalling molecule made from anthranilate and a beta-keto fatty acid.
Justification: GO has the molecular function GO:0102164 2-heptyl-3-hydroxy-4(1H)-quinolone synthase activity but no process term for quinolone-signal biosynthesis. Without one, the genes that supply this pathway - phnAB for anthranilate, the pqsABCDE operon, pqsH - can only be annotated to the downstream phenazine process they induce, which is what produced the over-annotation reviewed here.
Parent term: secondary metabolite biosynthetic process
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000162 L-tryptophan biosynthetic process | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA from the PTHR43418 LUCA node PTN000230074 (149 leaves), a well-founded IBD with eight experimental seeds from plants, fungi and bacteria, including P. aeruginosa TrpG (P20576). The function being transferred - anthranilate synthase component II in tryptophan biosynthesis - is the ancestral role of the clade. Reason: Defensible but not this paralogue's physiological job. PhnAB can supply anthranilate for tryptophan: trpE deletion revertants acquire a non-feedback-inhibited anthranilate synthase that arises from PhnAB. But phnAB deletions are tryptophan prototrophs, and the dedicated tryptophan enzyme in PAO1 is TrpEG, which is the primary anthranilate synthase at low cell density. The term is correct as a capability and peripheral to the gene's role, so it is kept as non-core rather than removed. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN000230074 · PTN000230074 (PTHR43418 LUCA node) SUPPORTS TRANSFER Eight seeds across three kingdoms; the ancestral role of the family is anthranilate synthase component II in tryptophan biosynthesis. UniProtKB:P20576 · P. aeruginosa TrpG SUPPORTS TRANSFER The organism's own tryptophan-pathway paralogue, experimentally annotated from the same 1990 paper; it is the gene that does this job in PAO1. Supporting Evidence: PMID:2153661 The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. file:PSEAE/phnB/phnB-uniprot.txt Double phnA-phnB deletions are tryptophan prototrophs; they make less PQS |
| GO:0002047 phenazine biosynthetic process | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from PTN000230115, a Gammaproteobacteria node of PTHR43418 whose seven leaves are the TrpG proteins of H. influenzae, Shewanella, Vibrio and Yersinia, the bifunctional TrpGD of E. coli and Salmonella, and phnB. The IBD is sole-seeded by this gene's PseudoCAP IMP row. Reason: PhnAB makes anthranilate, which is the precursor of the quinolone signal PQS; PQS then induces the phenazine operons. The phenazine ring is built from chorismate by PhzE via 2-amino-2-deoxyisochorismate and never passes through anthranilate, so PhnB performs no step of phenazine biosynthesis. Propagating the term is worse still: six of the seven leaves are enteric and other TrpG proteins from organisms that make no phenazines. Propagation Review Root cause: PROPAGATION BAD Failure modes: ROLE CONFLATION LINEAGE OR TAXON MISMATCH Sources checked: PANTHER:PTN000230115 · PTN000230115 SOURCE BAD A Gammaproteobacteria TrpG/TrpGD clade. Phenazine production is a lineage-specific trait of pseudomonads and a few other taxa, so this node cannot carry a phenazine term; 267 GOA rows now do (7 GO_Central, 260 TreeGrafter). UniProtKB:P09786 · phnB (PA1002) SOURCE BAD Sole seed. The seeding evidence is a pigment phenotype that acts through anthranilate supply to PQS, not through the phenazine pathway. Supporting Evidence: PMID:23449919 At the time it was believed that PhnAB generated anthranilate for pyocyanin production, but later work determined that anthranilate is instead a precursor for PQS production, which is required to induce genes for pyocyanin biosynthesis PMID:21454481 PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. |
| GO:0002047 phenazine biosynthetic process | IMP PMID:2153661 Identification and characterization of genes for a second an... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IMP, the sole seed of the PTN000230115 IBD. Essar et al. inactivated the phn genes and found reduced pyocyanin, and Swiss-Prot records the phnB disruption phenotype as decreased pyocyanine synthesis. The reduction is partial: phnA mutants retain low-level production, which is abolished only when trpE is also inactivated. Reason: A real phenotype, but an indirect one. The gene product's reaction is anthranilate synthesis; anthranilate feeds PQS synthesis, and PQS induces the phenazine operons. Nothing in the phenazine pathway consumes anthranilate. The accurate annotations for this gene are the anthranilate synthase activity (already present) and a quinolone-signal biosynthetic process, for which GO has no term (proposed below). Supporting Evidence: PMID:2153661 phnA mutants retain a low level of pyocyanin production. file:PSEAE/phnB/phnB-uniprot.txt anthranilate, a precursor for Pseudomonas quinolone signal PMID:23449919 No detectable PQS was produced by Δ phnAB , indicating that PhnAB is the anthranilate synthase responsible for PQS production in the presence of phenylalanine and tyrosine |
| GO:0004048 anthranilate phosphoribosyltransferase activity | IBA GO_REF:0000033 | REMOVE | Summary: IBA from PTN000230115, sole-seeded by E. coli P00904. That protein is the 531-residue bifunctional TrpGD, in which the phosphoribosyltransferase activity resides in the fused TrpD region. phnB is 200 residues and consists of a single glutamine amidotransferase domain (PROSITE PS51273, residues 2-195), with no TrpD region at all. Reason: A fusion-protein artefact. The activity cannot be carried out by a standalone TrpG subunit that lacks the phosphoribosyltransferase domain entirely; this is a domain-architecture mismatch rather than a judgment about the seed, whose own annotation is correct. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000230115 · PTN000230115 SOURCE BAD The node mixes TrpGD fusions with standalone TrpG subunits, so an IBD for a TrpD activity placed here is transferred to proteins without that domain. 267 GOA rows carry it. UniProtKB:P00904 · E. coli TrpGD SUPPORTS SOURCE BUT NOT TARGET Correct for the bifunctional seed (531 aa, TrpG + TrpD); the activity belongs to the fused region, which the target does not have. Supporting Evidence: file:PSEAE/phnB/phnB-uniprot.txt /note="Glutamine amidotransferase type-1" file:PSEAE/phnB/phnB-uniprot.txt FT CHAIN 1..200 file:PSEAE/phnB/phnB-bioinformatics/RESULTS.md phnB is 200 residues and consists of a single glutamine amidotransferase domain |
| GO:0004049 anthranilate synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Mapping from the curated EC number and Rhea reaction on the UniProt entry. It agrees with the experimental row below. Reason: Core activity. As for the IMP row, the complete reaction is carried out by the PhnA-PhnB heterotetramer, with PhnB supplying the glutamine amidotransferase half-reaction, so contributes_to would be the more accurate qualifier. Supporting Evidence: file:PSEAE/phnB/phnB-uniprot.txt Reaction=chorismate + L-glutamine = anthranilate + pyruvate + L- |
| GO:0004049 anthranilate synthase activity | IMP PMID:2153661 Identification and characterization of genes for a second an... | ACCEPT | Summary: PseudoCAP IMP from Essar et al., who showed that the cloned phnA phnB pair complements E. coli trpE and trpE(G) mutants, i.e. that it supplies anthranilate synthase activity in vivo, and that the activity recovered in trpE-deletion revertants of P. aeruginosa is not feedback-inhibited by tryptophan, as expected for PhnAB. Reason: Core molecular function of the gene product, supported by heterologous complementation of both anthranilate synthase components and by the curated catalytic triad and glutamine-binding residues. Supporting Evidence: PMID:2153661 The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. file:PSEAE/phnB/phnB-uniprot.txt the glutamine-binding beta subunit |
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Download this section (compressed HTML)Q: Should the GO:0002047 IBD at PTN000230115 be removed, given that six of its seven leaves are TrpG/TrpGD proteins of organisms that make no phenazines and that the seed phenotype acts through PQS signalling?
Q: Should the GO:0004048 IBD at PTN000230115 be moved down to the TrpGD fusion clade, so that standalone TrpG subunits no longer inherit an activity whose domain they lack?
Q: Is anthranilate supplied by PhnAB channelled to PqsA, or does it mix with the kynurenine-derived pool? The two routes are regulated differently and dominate under different conditions.
Experiment: Reconstitute purified PhnA and PhnB and measure anthranilate synthase kinetics with glutamine and with ammonia, alongside a PhnB catalytic-triad variant (C83A). This would convert the current in vivo complementation evidence into a direct demonstration of the subunit's amidotransferase contribution, and would let the EC number be supported experimentally for the P. aeruginosa enzyme rather than by similarity to E. coli TrpG.
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