The Molecular Basis of the β-Ketoacyl-ACP Synthase FabH in Catalyzing C–C Bond Formation of Acetoacetyl-ACP

β-Ketoacyl-ACP synthases (KAS) catalyze carbon skeleton extension in numerous metabolic routes such as the fatty acid biosynthesis pathway (FAS), among which FabH is the only known member that links the initiation stage to the elongation cycle of type-II FAS (FAS-II) by catalyzing condensation between acetyl-CoA and malonyl-ACP for the first β-keto-ACP intermediate acetoacetyl-ACP formation. Here, we reveal the substrate selection and condensation mechanisms of FabH from Escherichia coli. We demonstrate that EcFabH binds CoA and ACP using distinct regions in an irreversible compulsory order. The malonyl moiety is then delivered to a hydrophobic cage near the catalytic triad residues through front and middle door residues in the tunnel, and the substrate length is selected by a backdoor residue Phe87, ensuring the preferential recognition of EcFabH on acetyl moiety carried by CoA rather than longer substrates. Moreover, the malonyl moiety is locked in the cage by the acetylated Cys112 from the transacylation reaction, triggering the subsequent decarboxylation and condensation catalysis. Our study provides fundamental mechanistic insights into the initial extension of carbon skeletons catalyzed by FabH and homologues in FAS, PKS, and biotin biosynthesis pathways and may facilitate protein engineering and optimization for synthetic biological and pharmaceutical industry, as well as antibacterial drug development.