Structural and Kinetic studies of Acyl-CoA Carboxylases in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis is a pathogen responsible for around 10 million new TB cases and 1.4 million deaths per year, making it the primary cause of death from an infectious agent. A range of effective treatments are available, typically involving complex and lengthy protocols. Difficulties in treatment can arise with incorrect prescription and adherence to these protocols alongside the emergence of drug-resistant TB. This has generated a pressing need for improved TB therapies. The cell wall of M. tuberculosis is a complex structure with a diverse array of unique molecules such as mycolic acids. This complexity of lipids can provide M. tuberculosis with increased virulence capacity, but also resistance to antibiotics. Lipid metabolism pathways used to synthesise the unique mycolic acid components have become attractive therapeutic targets, with acyl-CoA carboxylase (YCC) enzymes functioning at the committed step of their biosynthesis. YCC enzymes are composed of biotin carboxylase-biotin carboxyl carrier protein (α) and carboxyltransferase (β) subunits. So far, whole complex structures are yet to be determined. This information could be essential to future drug design. This study is focussed on structural and kinetic characterisation of subunits relevant to mycolic acid synthesis, some of which include AccA3 (α), AccD5 (β) and AccE5 (ε) together forming an enzyme complex, the latter subunit proposed to improve complex stability. Co-expression and co-purification strategies were used to isolate our protein complex. Negative stain electron microscopy and kinetic bioassays helped uncover structural and kinetic properties of this enzyme complex. With the assistance of a crosslinking reagent, the purified acyl-CoA carboxylase AccA3-AccD5-AccE5 was shown to be catalytically active in vitro but did not assemble into a complex that was viable for further imaging studies beyond negative stain electron microscopy screening. Possible explanations for this are discussed. Future studies could employ the use of substrate addition, more variables involved in the crosslinking procedure and increasing AccE5 expression levels in order to overcome limitations shown when attempting to achieve a stable complex. Further to this, we have demonstrated the potential for smaller complexes to be catalytically active which could have implications for future design of therapeutics, as shall be discussed.