Mechanism-based crosslinking probes capture E. coli ketosynthase FabB in conformationally-distinct catalytic states.

Ketosynthases (KS) catalyse essential carbon-carbon bond forming reactions in fatty acid biosynthesis using a two-step, ping-pong reaction mechanism. In E. coli, there are two homodimeric elongating KSs, FabB and FabF, both of which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 14 carbons, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, we solved two crosslinked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty acid crosslinking probes that approximate catalytic steps. Both homodimeric structures possess asymmetric substrate binding pockets, suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active site gating residue, F392, potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based crosslinking methods to capture and elucidate at near atomic resolution conformational transitions accompanying KS-mediated catalysis. SynopsisCrystal structures of KS-ACP crosslinked complex elucidate chain length preference and substrate processing mechanism of E. coli FabB.