Abstract
Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys487 resulted in ∼95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys172 and Ala302 to be involved in the binding of ATP and Ser404 in the binding of CoenzymeA. The influence of mutations of the residues Val209 and Trp377 emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C26/C24 fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys172, Ala302 and Lys487 and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs.
Highlights
Mycobacterium tuberculosis (M.tb), an intracellular pathogen, is exquisitely adapted for human parasitization [1]
Discussion mymA is an important operon of M.tb due to its involvement in the remodeling of the cellular envelope under stressful conditions and in the intracellular survival of the pathogen [8,9,10]
Fatty Acyl-CoA Synthetase inhibitor Triacsin C has been widely studied as a therapeutic agent for the treatment of artherosclerosis and certain kind of tumors [35,36]
Summary
Mycobacterium tuberculosis (M.tb), an intracellular pathogen, is exquisitely adapted for human parasitization [1]. The pathogen resides in the host macrophages, where it encounters various stressful conditions such as changes in pH, exposure to reactive oxygen, nitrogen intermediates, degradative enzymes and deprivation of essential nutrients [5] During these conditions, the lipid rich cell surface of M.tb is often subjected to damage by the host assault. The lipid rich cell surface of M.tb is often subjected to damage by the host assault This pathogen has developed a variety of means to modify its cell envelope [6] for its survival in the hostile environment, emphasizing the importance of its cell envelope constituents as targets for the development of new antitubercular drugs. To gain further insight into the functioning of mymA operon, a potential target for developing antitubercular drugs, it is necessary to characterize its gene products
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