Substrate promiscuity of β‐Ketoacyl ACP Synthase III (KASIII): Understanding the structural basis for functional diversity of KASIII enzymes

β‐Ketoacyl ACP Synthase III (KASIII) catalyzes the Claisen condensation reaction that initiates fatty acid synthesis in bacteria and plants. KASIII from diverse sources can exhibit a broad range of substrate specificities. For example, E. coli KASIII uses straight chain acyl‐CoA substrates, whereas the B. subtilis homologs, KASIIIa and KASIIIb, use both straight and branched chain acyl‐CoA substrates. The structural basis for KASIII functional diversity remains obscure. Based on the hypothesis that the KASIII substrate specificity is determined by the rotamer conformation of a conserved Phe residue, we mutated several residues which are located close to this residue and can affect its rotamer conformation. The interactions of the mutated enzymes with their acyl‐CoA substrates (either straight or branched chain) were studied using Saturation Transfer Difference NMR. STD NMR data indicate that mutations of two residues in E. coli KASIII to the corresponding residues in B. subtilis KASIIIa increase straight and branched substrate binding by ~80% and ~66%, respectively. The corollary mutations in B. subtilis KASIIIa reduced the binding affinity for these substrates. Hence, this study has identified two key residues that govern the KASIII substrate specificity. This knowledge will enable engineering of KASIII biocatalysts with novel substrate specificities, which may have numerous downstream applications.