Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine Nε-acyltransferase involved in mycobactin biosynthesis

Abstract

Mycobactin acylation plays a crucial role in the ability of Mycobacterium tuberculosis to acquire intracellular iron during infection. M. tuberculosis Rv1347c, the lysine N ε-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 ε-amino group of lysine.