Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine N^ε-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^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 His¹³⁰ and Asp¹⁶⁸ indicated that both residues are critical for acyltransferase activity and suggests that His¹³⁰ is responsible for general base activation of the ε-amino group of lysine.