α-Isopropylmalate synthase (α-IPMS) is responsible for catalysing the first committed step in leucine biosynthesis. This pathway is found in plants and microorganisms, including pathogenic bacteria such as Mycobacterium tuberculosis and Neisseria meningitidis. α-IPMS catalyses a Claisen condensation reaction between α-ketoisovalerate (KIV) and acetyl coenzyme A (AcCoA) to form the product α-isopropylmalate (IPM). This enzyme undergoes feedback inhibition by the end product of the pathway, leucine. This regulation allows the control of the rate leucine biosynthesis. This project focuses on the α-IPMS enzymes from M. tuberculosis and N. meningitidis (MtuIPMS and NmeIPMS). These α-IPMS enzymes are homodimeric in structure. Each monomer consists of a catalytic domain which comprises of a (β/α)8 barrel fold, two subdomains and a regulatory domain, to which the allosteric binding of the natural inhibitor leucine occurs. The mechanism by which the allosteric binding of leucine leads to a decrease in enzymatic activity is not yet fully understood. Citramalate synthase (CMS) is responsible for catalysing the first committed step of threonine-independent isoleucine biosynthesis. This enzyme is extremely similar to α-IPMS in both the reaction which it catalyses and the catalytic and regulatory domain structure. CMS catalyses a Claisen condensation reaction between pyruvate and AcCoA to produce citramalate (CM). CMS is also feedback inhibited by the end product of its pathway, isoleucine. The similarity between α-IPMS and CMS enzymes resulted in and examination of the inhibitor selectivity of MtuIPMS. Amino acids in the leucine binding site were altered to their counterparts in the isoleucine binding site of the CMS enzyme to see if the selectivity of the leucine binding site could be interchanged. Results from this study show that it is possible to change inhibitor selectivity with a single amino acid substitution. However, changing the selectivity from leucine to isoleucine was unsuccessful. Instead, one of the MtuIPMS variants displayed significantly increased sensitivity to an alternative amino acid, norvaline. The MtuIPMS variants were expressed and purified using immobilised metal affinity chromatography and size-exclusion chromatography. These variants were then kinetically characterised and displayed similar binding affinities and turnover rates for the natural substrates to the wild-type enzyme. As expected changes to the leucine binding pocket had drastic effects on the sensitivity of the enzyme to its natural inhibitor. This work is described in Chapter 2 of this thesis. The mechanism by which the regulatory signal is transferred from the allosteric leucine binding site to the catalytic site in α-IPMS is not fully understood. NmeIPMS variants were created based on preliminary molecular dynamic simulations which indicated that significant changes in residue contacts were associated with leucine binding. Chapter 3 describes studies that explore the effect of single amino acid substitutions of NmeIPMS. The NmeIPMS variants were expressed and purified similarly to MtuIPMS, using immobilised metal affinity chromatography and size-exclusion chromatography. Variants were subsequently characterised via mass spectrometry, differential scanning fluorimetry and kinetic assays. It was found that each variant generated retained sensitivity to leucine but displayed significant differences in the catalytic efficiencies with AcCoA. One of the generated variants also displayed a significant increase in thermal stability. Results are drawn together in Chapter 4 along with future directions of this research. This chapter details knowledge gained into protein structure and allosteric mechanisms in this thesis.

α-Isopropylmalate synthase (α-IPMS) catalyses the first committed step in leucine biosynthesis in bacteria, including Neisseria meningitidis and Mycobacterium tuberculosis. It catalyses the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate (α-IPM). Like many key enzymes in biosynthesis, α-IPMS is inhibited by the end-product of the biosynthetic pathway, in this case leucine. α-IPMS is homodimeric, with monomers consisting of a (β/α)8-barrel catalytic domain, two subdomains and a C-terminal regulatory domain, responsible for binding leucine and providing feedback inhibition for leucine biosynthesis. The exact mechanism of feedback inhibition in this enzyme is unknown, despite the elucidation of crystal structures with and without leucine bound. This thesis explores the nature of allosteric regulation in α-IPMS, including the effects of the regulatory domain and the importance of structural asymmetry on catalytic activity. Chapter 2 details the characterisation of wild-type α-IPMS from N. meningitidis (NmeIPMS). This protein was successfully cloned, expressed and purified by metal-affinity and size-exclusion chromatography. NmeIPMS has similar characteristics to previously characterised α-IPMSs, being a dimer and demonstrating substrate binding affinities in the micromolar range. This enzyme has a turnover number of 13s⁻¹ and is sensitive to mixed, non-competitive inhibition by the amino acid leucine. Small angle X-ray scattering experiments reveal that the solution-phase structure of this protein is likely similar to existing crystal structures of other α-IPMSs. In Chapter 3, substitutions of residues potentially involved in the binding and transmission of the leucine regulatory mechanism are described. Most of these amino acid substituted variants reduce enzyme sensitivity to leucine, and one variant is almost entirely insensitive to this inhibitor. Another of these variants demonstrates an unexpected decrease in substrate affinity, despite the substituted residue being located far from the active site. The independence of α-IPMS domains is investigated in Chapter 4. The catalytic domains were isolated from NmeIPMS and the α-IPMS from M. tuberculosis (MtuIPMS), and found to be unable to catalyse the condensation of substrates, despite maintaining the wild-type structural fold. Complementation studies with Escherichia coli cells lacking the gene for α-IPMS show that the truncated variants are unable to rescue growth in these cells. Binding of α-KIV in the truncated NmeIPMS variant is much stronger than in the wild-type, and this may be the reason for lack of competent catalysis. A crystal structure was solved for the truncated variant of NmeIPMS and indicates that the regulatory domain is required for proper positioning of large regions of the protein. Two isolated regulatory domains from NmeIPMS were cloned, but with limited success in characterisation. Finally, Chapter 5 describes substitutions made in MtuIPMS to affect relative domain orientations within the protein. Dimer asymmetry is investigated by substituting residues at the domain interfaces. These substitutions did have some effect on catalysis and inhibition, but did not show any change in average solution-phase structure. These results are drawn together in the greater context of allostery in general in Chapter 6, along with ideas for future research in this field. This chapter reviews the insights gained into protein structure from this thesis, particularly the importance of residues at protein domain interfaces. The asymmetry in the α-IPMS structure is discussed, along with small-molecule binding regulatory domains.