Removal of the C-Terminal Regulatory Domain of α-Isopropylmalate Synthase Disrupts Functional Substrate Binding Frances H. A. Huisman, † Nayden Koon, ‡ Esther M. M. Bulloch, ‡ Heather M. Baker, ‡ Edward N. Baker, ‡ Christopher J. Squire,* ,‡ and Emily J. Parker* ,† † Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, Christchurch, New Zealand ‡ Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland, New Zealand * S Supporting Information ABSTRACT: α-Isopropylmalate synthase (α-IPMS) catalyzes the metal-dependent aldol reaction between α-ketoisovalerate (α-KIV) and acetyl-coenzyme A (AcCoA) to give α- isopropylmalate (α-IPM). This reaction is the first committed step in the biosynthesis of leucine in bacteria. α-IPMS is homodimeric, with monomers consisting of (β/α) 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 α-IPMS enzymes of both Neisseria meningitidis (NmeIPMS) and Mycobacterium tuberculosis (MtuIPMS) results in enzymes that are unable to catalyze the formation of α-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 α-IPMS gene, where transformation with the plasmids encoding the truncated α-IPMS enzymes was not able to rescue α-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 α-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 α-KIV and AcCoA, they are still able to bind the substrate α-KIV. It is proposed that the regulatory domain is crucial for ensuring protein dynamics necessary for competent catalysis. O ne of the most fascinating aspects of enzymes is how exquisitely they are regulated within metabolic pathways. A key method of such regulation is the binding of pathway end products to specific allosteric binding sites located far from where catalysis takes place. Structural studies have shown that for some enzymes the distances between the binding sites for the allosteric inhibitor and substrates can be quite large, and that the allosteric ligand binding sites can be formed by distinct regulatory domains that are appended to the core catalytic structure. 1,2 In some cases, the regulatory domain acts quite independently of the active site and may be removed to give an unregulated enzyme. 3,4 What are harder to investigate, however, are enzymes for which the removal of a structurally distinct regulatory domain dramatically alters catalytic activity. For these enzymes, contributions to catalysis of this regulatory architecture need to be better understood. One such allosterically regulated enzyme is α-isopropylmalate synthase (α-IPMS, EC 2.3.3.13), a member of the Claisen condensing family. α-IPMS catalyzes the condensation of α- ketoisovalerate (α-KIV) and acetyl-coenzyme A (AcCoA) to form (S)-α-isopropylmalate [α-IPM (Figure 1)]. This is the first committed step in the biosynthesis of leucine, and the enzyme is feedback-inhibited by this amino acid, which binds to an allosteric regulatory domain. 5,6 Most characterized α-IPMS enzymes are homodimeric, 7-9 although some may adopt a tetrameric form. 5,10 All α-IPMS enzymes are reported to be dependent on a divalent metal ion for full activity, and some also require a monovalent cation. 11 Crystal structures of α-IPMS from Mycobacterium tuberculosis (MtuIPMS) have defined the binding sites of α-KIV and leucine, but the leucine-bound and leucine-free structures overlay so closely that it is not possible to deduce how effector binding may inhibit catalysis. This protein is a homodimer of 70 kDa monomers, each with an (β/α) 8 barrel catalytic domain and a βαβ sandwich regulatory domain. 8 Two subdomains and a flexible linker region separate the catalytic and regula- tory domains (Figure 2). α-KIV and a Zn 2+ ion bind at the C-terminal end of the (β/α) 8 barrel, and leucine binds at the Received: November 16, 2011 Revised: February 3, 2012 Published: February 21, 2012 Figure 1. Reaction catalyzed by α-IPMS. Article pubs.acs.org/biochemistry © 2012 American Chemical Society 2289 dx.doi.org/10.1021/bi201717j | Biochemistry 2012, 51, 2289-2297