fabY (PA5174, Q9HU15) notes

Deep research (just deep-research) was not available in this session (no provider API keys),
so no -deep-research-<provider>.md file exists. Literature was researched directly: the
cached publications, PubMed searches ("FabY Pseudomonas aeruginosa PA5174", "FabY
beta-ketoacyl synthase Pseudomonas"; only PMID:22753059 and PMID:22753057 are returned), and
the PMC full text of PMID:22753059, which was upgraded in the publications cache from abstract
to full text (PMC3457228) for this review. The GONUTS wiki page
(https://gowiki.tamu.edu/wiki/index.php/PMID:22753059) returned only a maintenance notice
("site temporarily offline"), so the CACAO students' own evidence notes could not be read.

Identity

• 634-aa KASI/II-family (KS3 domain) condensing enzyme; active-site Cys281 (UniProt FT).
• FabY class defined by Yuan et al. 2012: "We propose distinguishing β-ketoacyl-ACP synthases
  with KASI/II domains and a preference for short-chain acyl-CoA acceptor substrates as the new
  FabY class of condensing enzymes" PMID:22753059.
• Restricted distribution: "FabY is also more narrowly distributed at the species level"
  PMID:22753059 (P. aeruginosa, P. fulva, P. mendocina, Azotobacter vinelandii).
• Crystal structure 4CW4 (1.35 A), homodimer; FabY is a "noncanonical fatty acid biosynthesis
  starter enzyme" whose insertions resemble fungal FAS scaffolding [PMID:25456814, abstract only].

Function (PMID:22753059, full text)

• Genetic: P. aeruginosa cosmids rescuing a FabH-depleted E. coli all began just upstream of
  PA5174; "PA5174 is thus by itself sufficient to complement FabH and restore initiation of FAS";
  plasmid PA5174 allowed deletion of E. coli fabH: "Growth analysis confirmed complete
  fabH-PA5174 cross complementation" (Fig. 3).
• Deleting all four KASIII homologs (PA3333, PA3286, PqsC, PqsD) had no growth phenotype (Fig. 2C).
• ΔPA5174: "doubling time increased from 19 to 62 min in the absence of PA5174" (Fig. 4A);
  [3H]acetate labeling: "the larger decrease in the rate of label incorporation for FAS in
  comparison to RNA indicates particular attenuation specific to FAS" (Fig. 4B).
• Enzymology (Fig. 8): "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";
  product reduced by FabG/FabA/FabI to butyryl-ACP; "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"; "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".
• Downstream phenotypes of ΔPA5174 (all consequences of reduced acyl-ACP supply):
• rhl QS: "The amount of acyl-HSL in the ΔPA5174 strain was nearly undetectable in comparison
  to that in the wild type" (Fig. 5A, TLC + C. violaceum overlay); rhamnolipid and swarming loss.
• las: "The ΔPA5174 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" (Fig. 6A); elastase lost.
• PQS: "The PQS level was reduced to a trace in the supernatant extract when PA5174 was
  deleted" (Fig. 6C); pyocyanin lost.
• Siderophore: CAS plate, "the ΔPA5174 strain did not produce a zone, indicating a lack of
  relative siderophore production" (Fig. 7). Authors' rationale: "Since pyoverdine is
  coregulated by QS systems (79) and is assembled on a myristate fatty acid residue (21, 34)".
• Authors' interpretation: "The progression of QS in the ΔFabY mutant is likely delayed by the
  lack of fatty acid precursors".

Bypass (PMID:22753057, abstract)

Exogenous ≥C8 fatty acids rescue ΔfabY via the KASIII enzyme PA3286, which condenses C8-CoA with
malonyl-ACP; "PA3286 in P. aeruginosa PAO1 intercepts C(8)-CoA by condensation with malonyl-ACP"
PMID:22753057.

Lipid A (PMID:24145528, full text)

• "The majority of lipid A species in the Δ fabY mutant lacked a single secondary lauroyl group,
  resulting in hypoacylated lipid A." PMID:24145528
• Mechanism proposed is indirect precursor limitation: "It is likely that decreased flux of the
  FAS pathway in the Δ fabY strain limits cellular pools of the C 12:0 -ACP donor available for
  the secondary acyl transferases." and the authors "cannot rule out differences in gene
  regulation" PMID:24145528. Lipid A acyltransferases (PA0011, PA3242 LpxL-like) perform the step.

CACAO assessment summary

Pattern: one well-characterised metabolic enzyme whose knockout has pleiotropic downstream
metabolite phenotypes; each downstream product pathway became a BP annotation. This is the
"phenotype read as process" failure, in a metabolic-precursor variant (necessity via substrate
supply, not participation).