The
biosynthetic pathway of peptidoglycan is essential for Mycobacterium
tuberculosis. We report here the acetyltransferase
substrate specificity and catalytic mechanism of the bifunctional N-acetyltransferase/uridylyltransferase from M.
tuberculosis (GlmU). This enzyme is responsible for the final
two steps of the synthesis of UDP-N-acetylglucosamine,
which is an essential precursor of peptidoglycan, from glucosamine
1-phosphate, acetyl-coenzyme A, and uridine 5′-triphosphate.
GlmU utilizes ternary complex formation to transfer an acetyl from
acetyl-coenzyme A to glucosamine 1-phosphate to form N-acetylglucosamine 1-phosphate. Steady-state kinetic studies and
equilibrium binding experiments indicate that GlmU follows a steady-state
ordered kinetic mechanism, with acetyl-coenzyme A binding first, which
triggers a conformational change in GlmU, followed by glucosamine
1-phosphate binding. Coenzyme A is the last product to dissociate.
Chemistry is partially rate-limiting as indicated by pH–rate
studies and solvent kinetic isotope effects. A novel crystal structure
of a mimic of the Michaelis complex, with glucose 1-phosphate and
acetyl-coenzyme A, helps us to propose the residues involved in deprotonation
of glucosamine 1-phosphate and the loop movement that likely generates
the active site required for glucosamine 1-phosphate to bind. Together,
these results pave the way for the rational discovery of improved
inhibitors against M. tuberculosis GlmU, some of
which might become candidates for antibiotic discovery programs.