Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine Nepsilon-acyltransferase involved in mycobactin biosynthesis.
Mycobactin acylation plays a crucial role in the ability of Mycobacterium tuberculosis to acquire intracellular iron during infection. M. tuberculosis Rv1347c, the lysine N(epsilon)-acyltransferase responsible for mycobactin acylation, represents a valid target for the development of novel anti-tubercular agents. Here we investigate the substrate specificity of Rv1347c, evaluate its kinetic mechanism and probe the contributions of active-site residues to catalysis. Our results confirm that Rv1347c demonstrates a preference for longer acyl-chains and suggest that mycobactin acylation occurs subsequent to mycobactin core assembly. Steady-state bisubstrate kinetics and dead-end inhibitor studies support a random sequential kinetic mechanism. Analysis of the pH dependence of k(cat)/K(m) revealed the presence of two groups that must be deprotonated for efficient catalysis. Mutagenesis of His(130) and Asp(168) indicated that both residues are critical for acyltransferase activity and suggests that His(130) is responsible for general base activation of the epsilon-amino group of lysine.
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Brenda A
BA
Frankel
John S
JS
Blanchard
D000217:Acyltransferases / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry*; D003198:Computer Simulation; D004789:Enzyme Activation; D004795:Enzyme Stability; D008956:Models, Chemical; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010080:Oxazoles / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010080:Oxazoles; C018608:mycobactins; D000217:Acyltransferases; C531527:Rv1347c, Mycobacterium tuberculosis