Structural and functional studies of mycolic acid synthesis in Mycobacterium tuberculosis

Despite the containment of tuberculosis (TB) in the developed world through antibiotics, it remains a public health problem of global proportion. The problem is made worse by emerging strains of the tubercle bacillus (Mycobacterium tuberculosis) that have become resistant against one or several of today's antitubercular drugs. New and more effective treatment options are urgently needed to control TB. Since the time of Koch, M. tuberculosis and related organisms have been known for the thick, waxy envelope surrounding the bacterium. This cell envelope comprises lipid, polysaccharide and peptide components in a highly organised layered structure. We understand in part, how these components are made and assembled by the bacillus, but the understanding is incomplete in many respects. This proposal focuses on a component of the cell envelope called mycolic acids, which make up to 10% of the biomass of the tubercle bacillus and are essential for survival. Mycolic acids are a particular kind of fatty acids that comprise a characteristic long arm, the meromycolate chain, and a shorter arm, the alfa-alkyl chain. We are interested in clarifying the 3-dimensional atomic structures of several enzymes that form a part of the catalytic assembly line making mycolic acids. We are also interested to learn how these enzymes work with other components of the assembly line, and how this is regulated. We employ X-ray crystallography and a range of other experimental techniques, to give us details of the molecular structure of the enzymes in question, and of interactions taking place between them. Such information will provide a more complete understanding of mycolic acid synthesis and may, in the long term, aid the process of finding new therapeutics against this ancient adversary of humanity.

Mycolic acids are unusual fatty acids of up to 90 carbon atoms, which constitute an abundant and essential component of the unique cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis. Mycolic acids make up the outermost layer of the covalently linked mycolyl-arabinogalactan-peptidoglycan (mAGP) core of the mycobacterial cell envelope, which furnishes M. tuberculosis and related organisms with a highly impermeable barrier against bactericidal agents. In the past 2 decades, significant progress has been made towards unravelling the pathway of mycolic acid biosynthesis, but our knowledge of the structure and function of key enzymes is incomplete, and we know little of how pathway components are co-ordinated or regulated. This proposal focuses on the fatty acid synthase II complex, and one of its accessory enzymes, the 1700-residue polyketide synthase Pks13, which catalyses the final step of mycolic acid synthesis. Building on previous genetic and enzymological studies, we propose to employ a combination of X-ray crystallography, analytical ultracentrifugation and established activity assays, in order to a) fully characterise the structure of malonylCoA:ACP transacylase FabD in complex with substrates and inhibitors; b) elucidate the structure of the FabD-homologue FabD2 and characterise its enzymatic activity; c) determine the structure of the essential condensase Pks13; d) probe the hypothesis of FAS-II components forming specialised elongation complexes by verifying assembly states and mutual interactions in vitro; and e) elucidate the regulatory effect of Ser/Thr-phosphorylation of FabD and other FAS-II components. This project will help completing the 3D map of molecular structures of key enzyme of mycolic acid biosynthesis, provide a deeper understanding of mutual interactions of pathway components, and prime the development of inhibitors of the essential enzymes FabD and Pks13.