Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine Nepsilon-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(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.

journal_name

Arch Biochem Biophys

authors

Frankel BA,Blanchard JS

doi

10.1016/j.abb.2008.05.013

subject

Has Abstract

pub_date

2008-09-15 00:00:00

pages

259-66

issue

2

eissn

0003-9861

issn

1096-0384

pii

S0003-9861(08)00273-7

journal_volume

477

pub_type

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