Removal of the C-Terminal Regulatory Domain of α-Isopropylmalate Synthase Disrupts Functional Substrate Binding

alpha-Isopropylmalate synthase (alpha-IPMS) catalyzes the metal-dependent aldol reaction between alpha-ketoisovalerate (alpha-KIV) and acetyl-coenzyme A (AcCoA) to give alpha-Isopropylmalate (alpha-IPM). This reaction is the first committed step in the biosynthesis of leucine in bacteria. alpha-IPMS is homodimeric, with monomers consisting of (beta/alpha)(8) barrel catalytic domains fused to a C-terminal regulatory domain, responsible for binding leucine and providing feedback regulation for leucine biosynthesis. In these studies, we demonstrate that removal of the regulatory domain from the alpha-IPMS enzymes of both Neisseria meningitidis (NmeIPMS) and Mycobacterium tuberculosis (MtuIPMS) results in enzymes that are unable to catalyze the formation of alpha-IPM, although truncated NmeIPMS was still able to slowly hydrolyze AcCoA. The lack of catalytic activity of these truncation variants was confirmed by complementation studies with Escherichia coli cells lacking the a-IPMS gene, where transformation with the plasmids encoding the truncated alpha-IPMS enzymes was not able to rescue alpha-IPMS activity. X-ray crystal structures of both truncation variants reveal that both proteins are dimeric and that the catalytic sites of the proteins are intact, although the divalent metal ion that is thought to be responsible for activating substrate alpha-KIV is displaced slightly relative to its position in the substrate-bound, wild-type structure. Isothermal titration calorimetry and WaterLOGSY nuclear magnetic resonance experiments demonstrate that although these truncation variants are not able to catalyze the reaction between alpha-KIV and AcCoA, they are still able to bind the substrate alpha-KIV. It is proposed that the regulatory domain is crucial for ensuring protein dynamics necessary for competent catalysis.