PMID:22753057

Citation	Yuan, Y, Leeds, JA and Meredith, TC (2012) Pseudomonas aeruginosa directly shunts β-oxidation degradation intermediates into de novo fatty acid biosynthesis. J. Bacteriol. 194:5185-96
Abstract	We identified the fatty acid synthesis (FAS) initiation enzyme in Pseudomonas aeruginosa as FabY, a β-ketoacyl synthase KASI/II domain-containing enzyme that condenses acetyl coenzyme A (acetyl-CoA) with malonyl-acyl carrier protein (ACP) to make the FAS primer β-acetoacetyl-ACP in the accompanying article (Y. Yuan, M. Sachdeva, J. A. Leeds, and T. C. Meredith, J. Bacteriol. 194:5171-5184, 2012). Herein, we show that growth defects stemming from deletion of fabY can be suppressed by supplementation of the growth media with exogenous decanoate fatty acid, suggesting a compensatory mechanism. Fatty acids eight carbons or longer rescue growth by generating acyl coenzyme A (acyl-CoA) thioester β-oxidation degradation intermediates that are shunted into FAS downstream of FabY. Using a set of perdeuterated fatty acid feeding experiments, we show that the open reading frame PA3286 in P. aeruginosa PAO1 intercepts C(8)-CoA by condensation with malonyl-ACP to make the FAS intermediate β-keto decanoyl-ACP. This key intermediate can then be extended to supply all of the cellular fatty acid needs, including both unsaturated and saturated fatty acids, along with the 3-hydroxyl fatty acid acyl groups of lipopolysaccharide. Heterologous PA3286 expression in Escherichia coli likewise established the fatty acid shunt, and characterization of recombinant β-keto acyl synthase enzyme activity confirmed in vitro substrate specificity for medium-chain-length acyl CoA thioester acceptors. The potential for the PA3286 shunt in P. aeruginosa to curtail the efficacy of inhibitors targeting FabY, an enzyme required for FAS initiation in the absence of exogenous fatty acids, is discussed.
Links	PubMed PMC3457203 Online version:10.1128/JB.00860-12
Keywords	3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/classification; 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/metabolism; Cloning, Molecular; Escherichia coli/genetics; Escherichia coli/metabolism; Fatty Acids/biosynthesis; Fatty Acids/chemistry; Gene Expression Regulation, Bacterial/physiology; Genetic Complementation Test; Molecular Structure; Oxidation-Reduction; Pseudomonas aeruginosa/genetics; Pseudomonas aeruginosa/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
PSEAE:BEKAS		GO:0061990: beta-ketodecanoyl-[acyl-carrier-protein] synthase activity	ECO:0000315:		F		Figure 3: FAME mass spectra indicate a KASIII enzyme condenses C8-CoA with malonyl-ACP to make beta-keto-decanoyl-ACP Figure 4B: ΔfabY in the TMT16 strain (strain in which 4 KASIII domains were deleted) resulted in cell death. The combination of ΔfabY and ΔPA3286 resulted in similar numbers of CFU as that found in the ΔfabY and TMT16 strain. Figure 4C: ΔPA3286 fed perdeuterated C10 did not incorporate deuterated fatty acids and had a mass spectrum almost identical to that of ΔPA3286 fed regular C10. TMT44 (strain complemented with PA3286) had deuterated FAME peaks and a mass spectrum similar to that of the wildtype.	complete CACAO 9584
PSEAE:BEKAS		GO:0042758: long-chain fatty acid catabolic process	ECO:0000315:		P		Figure 5A: Wild-type and ΔPA3286 strains were fed long-chain fatty acids. Only the wild-type strain produced subsequent fatty acids where the terminal 7 carbon atom was fully substituted with deuterium.	complete CACAO 9597
PSEAE:BEKAS	enables	GO:0061990: beta-ketodecanoyl-[acyl-carrier-protein] synthase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete
PSEAE:BEKAS	involved_in	GO:0042758: long-chain fatty acid catabolic process	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete

References

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