The Mechanism of Acetyl Transfer Catalyzed by <i>Mycobacterium tuberculosis</i> GlmU

The biosynthetic pathway of peptidoglycan is essential for <i>Mycobacterium tuberculosis</i>. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional <i>N</i>-acetyltransferase/uridylyltransferase from <i>M. tuberculosis</i> (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP-<i>N</i>-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 <i>N</i>-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 <i>M. tuberculosis</i> GlmU, some of which might become candidates for antibiotic discovery programs