A Hidden Binding Pocket in the β- ketoacyl-ACP Synthase FabB

Assembly-line enzymes carry out multi-step synthesis of diverse metabolites by using a handful of catalytic motifs in which minor structural differences control substrate specificity and reaction order. Here we examine differences in substrate binding to FabB and FabF, the two {beta}-ketoacyl-ACP synthases (KSs) responsible for fatty acid elongation in Escherichia coli, by exploring a peculiar mutational effect. In FabB, a blocking mutation in the acyl binding pocket yields a shifted, but broad product profile, while in FabF, the same mutation disrupts the binding of acyl chains longer than eight carbons (C8). X-ray crystal structures of the FabB mutant provide an explanation: a second, previously unobserved binding pocket allows medium-to-long acyl chains ([≥] C8) to bind with an alternate conformation. Molecular simulations suggest that this pocket is more stable in FabB than in FabF, where mutations reduce the catalytic competency of longer chains instead of shifting them to the alternate pocket. Our findings indicate that homologous KSs differ not only in their primary binding sites but also in the availability of alternative binding modes that can buffer against mutational effects and enable functional diversification.