The enzymes of the Claisen-condensing family catalyse the condensation reaction between acetyl coenzyme A (AcCoA) and an α-ketoacid. The well-characterised members of this family include α-isopropylmalate synthase (α-IPMS) from the L-leucine biosynthetic pathway, malate synthase (MS) in the glyoxylate pathway, citramalate synthase (CMS) from the threonine- independent L-isoleucine biosynthetic pathway, citrate synthase (CS) from the Krebs cycle, and homocitrate synthase (HCS) in the α-aminoadipate pathway (L-lysine biosynthetic pathway).

α-IPMS, CMS, and HCS are the key enzymes that catalyse the first committed steps in L- leucine, L-isoleucine, and L-lysine biosynthesis in microorganisms, respectively, and their catalytic activities are feedback regulated by the end-product of the corresponding biosynthetic pathway. Each monomeric subunit of α-IPMS and CMS is comprised of a C-terminal catalytic domain, two subdomains (I and II), and an N-terminal regulatory domain responsible for allosteric inhibition, whereas HCS consists of only a catalytic domain and two subdomains and lacks a discrete regulatory domain. In addition to the structural similarities, α-IPMS, CMS, and HCS share common reaction chemistry, which is a metal ion-dependent transfer of an acetyl group from AcCoA to an α-keto acid. This information, in addition to sequence similarities, indicates that these enzymes are evolutionarily related.

This thesis aimed to characterise the functionality of α-IPMS, CMS, and HCS to provide greater detail of the mechanisms by which they catalyse their reactions and to help develop a greater understanding of their evolutionary relationships and allosteric regulation.

Chapter 2 demonstrates the expression and biochemical characterisation of the wild-type HCS from Schizosaccharomyces pombe (SpoHCS) and a detailed description of its functionality in different environments. SpoHCS was successfully expressed and purified using immobilised metal ion affinity chromatography followed by size-exclusion chromatography. The enzyme was found to be tetrameric in solution, which is in contrast to the previously described oligomeric state for this enzyme. The kinetic characteristics of the wild-type SpoHCS were studied, in which the apparent KM values for the substrates were determined to be in the micromolar range with a turnover number of 4.2 ± 0.1 s-1, and catalysis was inhibited by L- lysine. The enzyme kinetic data showed a dependency on pH, temperature, and solution viscosity. The crystal structure of the enzyme was solved, which showed similar characteristics to previously reported crystal structures.

In Chapter 3, the influence of the accessory domain in the functionality of SpoHCS is described, which involved the substitution of residues predicted to be important in conformational stability and flexibility of the enzyme. The residue substitutions significantly affected the protein fold of the enzyme and its thermal stability. The majority of the mutated variants abolished enzymatic activity, and only three variants remained active; however, the sensitivity of the enzyme towards its substrates was significantly reduced. The kinetics and crystal structures of constructed variants show that the substituted residues play a crucial role in conformational stability and flexibility of subdomain I, which is proposed to be necessary for the enzyme functions.

Chapter 4 details the catalytic and regulatory responses of the α-IPMS from Neisseria meningitidis (NmeIPMS) to different sizes of protein domains (TRX and GST) added to the N- terminus of the regulatory domain. NmeIPMS was successfully cloned and expressed with an N-terminal TRX-tag and GST-tag; however, the purified NmeIPMS-GST was found to be aggregated. Both of the constructed NmeIPMS variants were catalytically active, but no allosteric inhibition was observed in the presence of L-leucine. However, both isothermal titration calorimetry and differential scanning fluorimetry data showed that L-leucine was still bound to NmeIPMS-TRX. Small-angle X-ray scattering experiments were performed for both the wild-type enzyme and NmeIPMS-TRX to characterise their solution-phase structures and any conformational changes that occurred in the presence of L-leucine. However, both enzymes were found to aggregate in the presence of L-leucine.

Chapter 5 describes the inhibitor selectivity of the α-IPMS from Mycobacterium tuberculosis (MtuIPMS), an enzyme that catalyses the condensation of AcCoA and α-KIV as the primary step in the biosynthesis of L-leucine and is allosterically regulated by L-leucine. Key residues predicted to be involved in L-leucine binding were rationally substituted and the allosteric inhibition and inhibitor selectivity of the enzyme variants were studied. Kinetic assays demonstrated that all variants were catalytically active. The inhibition studies showed that Ala567 and Ile627 residues in the regulatory domain of MtuIPMS significantly influence the specificity of the enzyme towards allosteric ligands, so that the substitutions of Ala567 and Ile627 for valine and alanine, respectively, altered the inhibition specificity of the enzyme. In addition, inhibition data illustrated that L-leucine inhibition for MtuIPMS switched to L- isoleucine by the simultaneous substitutions of A567V/I627A. Furthermore, ITC data showed that the potential allosteric inhibitors bound to the enzyme variants, suggesting the substitutions had influenced the communication between allosteric and active site.

Chapter 6 investigates the physical characteristics and functionality of CMS from Methanococcus jannaschii (MjaCMS). The enzyme was expressed and purified, and its physical features were characterised, in which analytical gel filtration data showed that the enzyme was aggregated. However, kinetic characterisation showed that the enzyme was catalytically active with an apparent KM value of 74 ± 5 µM for pyruvate and 43 ± 2 µM for AcCoA, and a turnover number of 1.15 ± 0.05 s-1. The catalytic activity of the enzyme was found to be sensitive to non-competitive inhibition by L-isoleucine with Ki values of 166 ± 9 μM and 152 ± 11 μM for pyruvate and AcCoA, respectively.