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Abstract

Mycobacterium tuberculosis, one of the deadliest human pathogens, causes several million new infections and about two million fatalities annually. The cell wall of M.tuberculosis is endowed with a highly impermeable, complex array of diverse lipids such as mycolic acids, which bestow the bacterium with not only virulence, but also resistance to host immunity and antibiotics. Mycobacterial lipid metabolism has thus emerged as an attractive target for the design and development of novel anti-mycobacterial therapeutics. The first committed step in the biosynthesis of long-chain fatty acids – the carboxylation of acetyl-CoA to malonyl-CoA - is catalysed by the multi-functional multi-subunit acetyl-CoA carboxylase (ACC) enzyme. Some ACC complexes, especially from actinobacteria, are active on diverse substrates and are generally referred to as acyl-CoA carboxylases (YCCs). Typically, YCCs are composed of biotin carboxylase, biotin carboxyl carrier protein (collectively known as alpha) and carboxyltransferase (beta) subunits. Interestingly, the genomes of most mycobacteria code for three alpha subunits (AccA1 - AccA3), six beta subunits (AccD1 - AccD6), and a unique epsilon subunit (AccE5) while most other forms of life possess not more than two YCCs. Despite the significant roles of YCCs in mycobacterial fatty and mycolic acid biosyntheses and hence in cell wall integrity and antibiotic resistance, a comprehensive understanding of their properties and functions is lacking.

This dissertation is focused on the structural and functional characterisation of the essential components (AccA3, AccD4 - AccD6, and AccE5) of M. tuberculosis YCCs implicated or known to be involved in fatty acid metabolism. X-ray crystallography and complementary biophysical and biochemical approaches have been employed in an attempt to address questions concerning interactions between YCC components and differences in beta subunit substrate specificity.

Multiple co-expression and co-purification strategies yielded YCC complexes (the propionyl-CoA carboxylase AccA3-AccD5 and the putative long-chain acyl-CoA carboxylase AccA3-AccD4) that were catalytically active but did not assemble into stable forms amenable to structural analyses; possible explanations for these observations have been discussed in detail. Significant effort was invested in the production of the epsilon subunit AccE5, but its association with its interacting partners (AccA3 and AccD5) could not be investigated due to technical impediments stemming, presumably, from the intrinsic disordered nature of the protein.

Biophysical studies of AccD4 and AccD6 (the beta subunit of ACC) revealed unexpected structural diversity in the M. tuberculosis YCC beta subunit subfamily. Unlike all other actinobacterial homohexameric beta subunits characterised to date, AccD4 and AccD6 were found to function as lower oligomers, highlighting that hexameric assembly is not a requisite for carboxyltransferase function. Endeavours to crystallize AccD4, in apo-form or in complex with substrate/cofactor analogs, were unsuccessful. The high-resolution crystal structure of AccD6, on the other hand, was determined by the method of molecular replacement. The structure of AccD6 has elucidated the molecular basis of homodimeric arrangement, besides throwing light on the conserved and non-conserved features of the active site, and the putative determinants of substrate specificity.

Taken together, the findings of this study have added to the existing knowledge of the M. tuberculosis structural proteome and have furthered our understanding of the biophysical attributes and functions of YCC beta subunits, validated anti-mycobacterial drug targets. Interesting insights into the likely molecular evolution of YCC beta subunits have been acquired.