Biochemical and structural studies of Mycobacterium tuberculosis isocitrate lyase

Abstract

The work described in this Thesis has focused on the study of Mycobacterium tuberculosis isocitrate lyase (ICL), a key enzyme of the glyoxylate cycle. The work included recombinant production and purification of the different ICL isoforms, the development of new biophysical assays to study ICL inhibitors, biochemical and structural studies of ICL isoform 2, and the sustainable synthesis of pharmaceutically important nitrogen-heterocycles using itaconic acid, a weak inhibitor of ICL, as a starting material. The use of the Escherichia coli and Mycobacterium smegmatis expression systems to produce the four isoforms of ICL (isoforms 1, 2, 2a and 2b) was explored. Soluble ICL1, ICL2 and ICL2b were successfully produced and purified. Recombinant ICL2, widely regarded as unstable was found to be a stable enzyme that can be stored in buffers without imidazole. A combined nuclear magnetic resonance (NMR) and thermal shift assay strategy was developed to study ICL inhibitors using ICL1 as a model system. The NMR-based activity assay enables the detection of substrate (isocitrate) consumption and product (succinate) formation in real time, allowing enzyme kinetics and enzyme inhibition to be followed and quantified. It is informative but relatively low throughput. In contrast, the thermal shift assay measures protein stability upon ligand binding. It is an indirect assay but allows the screening of ICL ligands in a high-throughput manner. The effectiveness of this combined strategy was demonstrated by using existing ICL1 inhibitors. Protein X-ray crystallography was used for the structural and biochemical characterisation of ICL2, which was found to be a tetramer. Each monomeric unit contains two distinct domains. Structural and kinetic studies revealed that the C-terminal domain is an acetyl-coenzyme A binding domain. Binding of acetyl-CoA leads to a global conformational change, which leads to the activation of the isocitrate lyase and methylisocitrate lyase activities of ICL2.The synthesis of pharmaceutically important nitrogen-heterocycles using itaconic acid, a weak inhibitor of ICL, was also investigated. A library of diverse N-heterocycles including dihydroindolone, indole, dihydroindolizinone, 2-pyridone, pyrido[1,2-a]indole dihydrocarbazolone and carbazole have been prepared from dimethyl itaconate and pyrrole, two compounds that are readily attainable from biomass.