dissecting the role of critical residues and substrate preference of a fatty acyl-coa synthetase (fadd13) of mycobacterium tuberculosis解剖临界残留的作用和衬底偏好的脂肪酰coa合成酶(fadd13)的结核分枝杆菌.pdfVIP

Dissecting the Role of Critical Residues and Substrate Preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis 1 1 1,2 1 3 1 Garima Khare , Vibha Gupta , Rakesh K. Gupta , Radhika Gupta , Rajiv Bhat , Anil K. Tyagi * 1 Department of Biochemistry, University of Delhi South Campus, New Delhi, India, 2 Department of Microbiology, Ram Lal Anand College, University of Delhi South Campus, New Delhi, India, 3 School of Biotechnology, Jawaharlal Nehru University, New Delhi, India 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 represent