FabY (PA5174) is the beta-ketoacyl-acyl carrier protein synthase that initiates type II fatty acid synthesis in Pseudomonas aeruginosa. Although it belongs to the KASI/II (FabB/FabF-like, ketosynthase KS3) family of elongation condensing enzymes, it functions as the cell's FabH equivalent, condensing acetyl-CoA with malonyl-ACP to give the primer beta-acetoacetyl-ACP; it also accepts butyryl- and hexanoyl-CoA but not longer acyl-CoAs, and does not efficiently elongate acyl-ACPs. FabY fully replaces FabH in Escherichia coli, whereas P. aeruginosa lacking all four KASIII homologs grows normally. The 634-residue soluble enzyme is a homodimer with N- and C-terminal extensions outside the KS domain, and is restricted to P. aeruginosa and a few close relatives. Deleting fabY slows growth and de novo fatty acid synthesis. Downstream, the mutant makes less of the fatty acid-derived products, including rhl acyl-homoserine lactones, PQS, rhamnolipids, siderophores and secondary lauroyl chains of lipid A, and becomes hypersusceptible to several antibiotics. Exogenous medium-chain fatty acids bypass FabY through the KASIII enzyme PA3286.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004315 3-oxoacyl-[acyl-carrier-protein] synthase activity | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic propagation of the elongating KASI/II condensing activity (acyl-ACP + malonyl-ACP, EC 2.3.1.41) from the PTN000918584 node seeded by E. coli FabB/FabF, M. tuberculosis KasA/KasB, Arabidopsis KAS and human OXSM. FabY sits in this clade by sequence but has diverged to the initiating reaction with an acyl-CoA acceptor. Reason: FabY is inside the KASI/II clade that inherited the elongating activity, so the node placement is reasonable for the family. FabY itself, though, has target-specific evidence of functional divergence. Purified FabY condenses acetyl-CoA (and C4/C6-acyl-CoA) with malonyl-ACP, the KASIII reaction (EC 2.3.1.180, GO:0033818). In a coupled FabG/FabA/FabI assay it gave no further elongation products, which the authors read as an inability to elongate acyl-ACP efficiently (PMID:22753059, Fig. 8B). GO:0033818 is a sibling of GO:0004315 under acyltransferase activity, not a child, so this is a lateral substrate correction to the term the gene already carries by IDA. The no-elongation result is one in-vitro negative, so a weak residual elongating activity is not excluded; this is raised in suggested_questions. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000918584 Β· PTN000918584 SUPPORTS SOURCE BUT NOT TARGET KASI/II elongating-condensation node seeded by FabB/FabF-type donors. It is sound for the clade, but FabY has diverged to an acyl-CoA acceptor (initiating, KASIII-type reaction). Proposed replacements: beta-ketoacyl-acyl-carrier-protein synthase III activity Supporting Evidence: PMID:22753059 The lack of additional bands indicates that PA5174 cannot efficiently carry out the next round of condensation for chain elongation using acyl-ACP as a substrate PMID:22753059 the specific activities of PA5174 and E. coli FabH with malonyl-ACP and acetyl-CoA as substrates were 13.4 and 7.4 pmol/min/ng under the same assay conditions |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: Cytosolic location propagated from E. coli FabB and mycobacterial KAS donors. Type II FAS condensing enzymes are soluble proteins acting on soluble ACP thioesters. Reason: FabY has no predicted transmembrane segment or signal peptide, and it was purified as a soluble His-tagged protein that is active on soluble malonyl-ACP and acetyl-CoA. Dissociated type II FAS enzymes work in the bacterial cytoplasm, so the inherited cytosol location fits the core activity. No direct localization assay exists for FabY. Supporting Evidence: PMID:22753059 Recombinant PA5174 was expressed in E. coli and purified by His tag affinity chromatography in order to characterize in vitro enzymatic activity. |
| GO:0006633 fatty acid biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: FabY catalyses the initiating condensation of type II fatty acid synthesis in P. aeruginosa. Q9HU15 appears among the IBA sources because its own experimental annotation helped seed the node. Reason: The process is the core role of the gene and is confirmed directly for Q9HU15. FabY complements E. coli fabH deletion. ΞfabY specifically lowers [3H]acetate incorporation into fatty acids, and the FabY product is extended by FabG/FabA/FabI (PMID:22753059). Q9HU15 in the WITH/FROM list reflects its own experimental grounding, not circularity. Supporting Evidence: PMID:22753059 PA5174 is thus by itself sufficient to complement FabH and restore initiation of FAS |
| GO:0006633 fatty acid biosynthetic process | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway mapping of the curated UniProt PATHWAY line (lipid metabolism; fatty acid biosynthesis). Reason: Consistent with the experimental evidence (PMID:22753059) and with the IBA and IMP rows for the same term. The curated UniProt function statement, backed by PubMed evidence, places FabY at the initiation step of fatty acid synthesis. Supporting Evidence: file:PSEAE/fabY/fabY-uniprot.txt Involved in the initiation of the fatty acid biosynthesis. |
| GO:0006633 fatty acid biosynthetic process | IMP PMID:22753059 Fatty acid biosynthesis in Pseudomonas aeruginosa is initiat... | ACCEPT | Summary: CACAO annotation. The cited paper deletes PA5174 in PAO1 (allelic exchange, strain TMT39). Growth slows (doubling time 19 to 62 min, Fig. 4A) and [3H]acetate incorporation into fatty acids falls more than [3H]uridine incorporation into RNA (Fig. 4B). Plasmid PA5174 or E. coli fabH restores both. Reason: The CACAO term matches what the paper shows. The macromolecular labeling experiment measures de novo fatty acid synthesis flux directly, and the defect is complemented. Biochemistry in the same paper (Fig. 8) shows FabY catalyses a step of the process, so this is participation, not just necessity. The granularity is right: GO has no "fatty acid synthesis initiation" term, and the initiating condensation is captured by the MF row. Supporting Evidence: PMID:22753059 However, the larger decrease in the rate of label incorporation for FAS in comparison to RNA indicates particular attenuation specific to FAS PMID:22753059 doubling time increased from 19 to 62 min in the absence of PA5174 |
| GO:0009245 lipid A biosynthetic process | IMP PMID:24145528 Deletion of the Ξ²-acetoacetyl synthase FabY in Pseudomonas a... | MARK AS OVER ANNOTATED | Summary: CACAO annotation. The cited paper shows by FAME analysis (Fig. 1), LPS SDS-PAGE (Fig. 2) and lipid A ESI-MS (Fig. 4, Table 2) that most lipid A in ΞfabY lacks one secondary lauroyl (C12:0/2-OH-C12:0) chain and carries extra L-Ara4N. Exogenous decanoate partly restores secondary acylation. Reason: The phenotype is real and complemented, but the term reads a downstream metabolite phenotype as process participation (phenotype read as process). FabY makes beta-acetoacetyl-ACP. The lipid A steps that are defective, secondary lauroylation, are carried out by the LpxL-like acyltransferases PA0011 and PA3242 using C12:0-ACP. The authors explain the defect as reduced FAS flux limiting the C12:0-ACP donor pool, and they say they cannot rule out a regulatory cause. FabY therefore supplies a precursor and performs no step of lipid A biosynthesis. Its role is fully covered by fatty acid biosynthetic process. Initiating condensing enzymes elsewhere, such as E. coli FabH, are not conventionally annotated to lipid A biosynthesis for the same reason. Not removed: this is an experimental row, and the precursor link is direct (all lipid A acyl chains come from FAS acyl-ACP). Supporting Evidence: PMID:24145528 It is likely that decreased flux of the FAS pathway in the Ξ fabY strain limits cellular pools of the C 12:0 -ACP donor available for the secondary acyl transferases. PMID:24145528 Although we certainly cannot rule out differences in gene regulation of either a secondary lipid A lauroyl transferase, a putative lauroyl deacylase, or another component of the multifaceted Ξ fabY phenotype to explain the hypoacylation |
| GO:0009372 quorum sensing | IMP PMID:22753059 Fatty acid biosynthesis in Pseudomonas aeruginosa is initiat... | MARK AS OVER ANNOTATED | Summary: CACAO annotation. ΞPA5174 makes almost no rhl acyl-HSL (TLC with C. violaceum overlay, Fig. 5A) and only a trace of PQS (Fig. 6C), with loss of rhamnolipid, swarming, elastase and pyocyanin. Las 3-oxo-acyl-HSL stays near wild type (Fig. 6A). Plasmid PA5174 restores, and even raises, signal levels. Reason: The paper shows muted QS signal output, but as a consequence of reduced fatty acid precursor supply, not because FabY acts in signalling (phenotype read as process). The authors predicted the rhl defect from the chemistry: C4-HSL is made by RhlI from butanoyl-ACP, which derives from the FabY product. They interpret the delay in QS as a lack of fatty acid precursors. The las signal is unaffected despite the FAS defect, which argues against a direct role. FabY neither synthesises nor senses a signal. Precursor supply into acyl-HSL and PQS synthesis is already captured by fatty acid biosynthetic process. The authors do note a correlation between FabY dosage and QS output that warrants study, so the row is not removed. Supporting Evidence: PMID:22753059 The progression of QS in the ΞFabY mutant is likely delayed by the lack of fatty acid precursors PMID:22753059 The ΞPA5174 mutant apparently can synthesize 3-oxo-acyl-HSL to wild-type levels in LB liquid culture after 24 h of incubation despite the FAS defect |
| GO:0016746 acyltransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the thiolase-like superfamily signature (IPR016039). Reason: Correct but far more general than the evidence. FabY's acyltransferase activity is the specific condensation of acetyl-CoA with malonyl-ACP (EC 2.3.1.180), shown for the purified enzyme (PMID:22753059). Refine to GO:0033818, a descendant of acyltransferase activity that the gene already carries by IDA. Proposed replacements: beta-ketoacyl-acyl-carrier-protein synthase III activity Supporting Evidence: file:PSEAE/fabY/fabY-uniprot.txt Catalyzes the condensation of acetyl coenzyme A (acetyl-CoA) with malonyl-acyl carrier protein (ACP) to make the fatty acid synthesis (FAS) primer beta-acetoacetyl-ACP. |
| GO:0019290 siderophore biosynthetic process | IMP PMID:22753059 Fatty acid biosynthesis in Pseudomonas aeruginosa is initiat... | MARK AS OVER ANNOTATED | Summary: CACAO annotation. On LB-chrome azurol S (CAS) plates, ΞPA5174 gives no Fe3+-sequestration halo, while wild type and the plasmid-complemented strain do (Fig. 7). This is the only siderophore assay in the paper. Reason: Too far downstream (phenotype read as process). A CAS halo reports secreted iron-chelating activity, so it cannot tell biosynthesis from regulation or secretion. The authors gave two possible causes: pyoverdine is assembled on a myristate residue, and it is co-regulated by quorum sensing, which is muted in this mutant. Neither was resolved, and pyoverdine was not quantified or structurally analysed. Even if the myristate route explains it, FabY only supplies a fatty acid precursor upstream of the nonribosomal peptide synthetase machinery and performs no step of siderophore biosynthesis. Kept rather than removed because the reduction is real and complemented. Supporting Evidence: PMID:22753059 the ΞPA5174 strain did not produce a zone, indicating a lack of relative siderophore production PMID:22753059 Since pyoverdine is coregulated by QS systems (79) and is assembled on a myristate fatty acid residue (21, 34) |
| GO:0033818 beta-ketoacyl-acyl-carrier-protein synthase III activity | IDA PMID:22753059 Fatty acid biosynthesis in Pseudomonas aeruginosa is initiat... | ACCEPT | Summary: CACAO annotation. Purified His-tagged PA5174 condenses acetyl-CoA with malonyl-ACP. The rate is 13.4 pmol/min/ng in a FabG-coupled NADPH assay, against 7.4 for E. coli FabH (Fig. 8A). The beta-acetoacetyl-ACP product is reduced by FabG/FabA/FabI to butyryl-ACP (urea-PAGE, Fig. 8B), and C2-C6 acyl-CoA acceptors are used but not C8-C16 (Fig. 8C). Reason: The CACAO term exactly matches the assayed reaction, EC 2.3.1.180 (acetyl-CoA + malonyl-ACP -> acetoacetyl-ACP + CoA + CO2), at the right granularity. GO:0033818 is defined by the reaction, not by KASIII fold membership, so it applies to this KASI/II-fold enzyme. This is the core activity of the gene. Supporting Evidence: PMID:22753059 the specific activities of PA5174 and E. coli FabH with malonyl-ACP and acetyl-CoA as substrates were 13.4 and 7.4 pmol/min/ng under the same assay conditions PMID:22753059 PA5174 utilized short-chain acyl-CoA as substrates, including acetyl-CoA, butyryl-CoA, and hexanoyl-CoA, but did not use any of the longer-chain acyl-CoA substrates |
| GO:0033818 beta-ketoacyl-acyl-carrier-protein synthase III activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated mapping from the curated UniProt catalytic activity (RHEA:12080, EC 2.3.1.180), which rests on PMID:22753059. Reason: Concordant with the IDA row. The underlying UniProt catalytic-activity annotation carries experimental evidence from PMID:22753059. Supporting Evidence: file:PSEAE/fabY/fabY-uniprot.txt Reaction=malonyl-[ACP] + acetyl-CoA + H(+) = 3-oxobutanoyl-[ACP] + CO2 + CoA |
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Download this section (compressed HTML)Q: Does FabY keep any measurable elongating KASI/II activity (acyl-ACP + malonyl-ACP), given that the conclusion that it does not rests on the absence of extra bands in one coupled urea-PAGE assay?
Q: What is the function of the ~200-residue N- and C-terminal extensions outside the KS domain, which resemble fungal FAS scaffolding elements? Do they determine acyl-CoA versus acyl-ACP acceptor selectivity?
Q: Is the reduced siderophore output of ΞfabY due to the pyoverdine myristate precursor or to quorum-sensing regulation? This decides whether any siderophore process term is warranted.
Q: How do Pseudomonas species that lack FabY (P. putida, P. fluorescens, P. syringae) initiate fatty acid synthesis?
Experiment: Steady-state kinetics of purified FabY with acetyl-CoA versus acetyl-ACP and C4-C10 acyl-ACP acceptors, to quantify any residual elongating activity and confirm acceptor selectivity.
Type: in vitro enzymology
Experiment: Quantify pyoverdine and pyochelin (fluorescence, HPLC) in ΞfabY with and without exogenous decanoate, and in a ΞfabY ΞrhlI background, to separate precursor-supply from QS-regulatory effects.
Type: metabolite quantification
Experiment: Truncation mutants lacking the N- or C-terminal extensions, tested for activity and dimerization, guided by the 4CW4 structure.
Type: structure-function mutagenesis
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