Craggs, Peter D.; (2019) Mechanistic characterisation and inhibitor identification of Mycobacterium tuberculosis bifunctional N-acetyltransferase/uridylyltransferase GlmU. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The mycobacterial cell envelope is a defining feature of the bacteria, primarily due to its highly-ordered, relatively impermeable nature that is likely one of the key attributes that has contributed to the success of this pathogenic over the last thousand years. Peptidoglycan is a unique and essential structural element that provides much of the strength and rigidity of the mycobacterial cell envelope. Most of the enzymes involved in the biosynthetic pathway of peptidoglycan have been shown to be essential for Mycobacterium tuberculosis growth. Mycobacterium tuberculosis GlmU is an essential bifunctional N-acetyltransferase, uridylyltransferase enzyme involved in the formation of uridine-diphosphate N-acetylglucosamine, which is the universal donor of N-acetylglucosamine for both peptidoglycan and lipopolysaccharide biosynthesis. This enzyme catalyses acetylation of glucosamine 1-phosphate, followed by uridylylation of N-acetylglucosamine 1-phosphate. Detailed characterisation of the kinetic mechanism ascertained that acetyl transfer progresses by the formation of a ternary complex, with acetyl coenzyme A binding preceding glucosamine 1-phosphate and coenzyme A the last product to dissociate. A novel ternary complex crystal structure, with glucose 1-phosphate and acetyl-coenzyme A, identified a candidate general base involved in the deprotonation of glucosamine 1-phosphate, as well as other important active site residues for substrate binding and catalysis. pH-rate studies and site-directed mutagenesis led to assignment of Histidine 374 as the catalytic general base. Solvent kinetic isotope effect experiments and pH-rate studies identified that acetyl transfer is partially rate-limiting. Small molecule screening led to the identification of novel inhibitors of GlmU catalysed acetyl transfer reaction. Inhibitors identified from the GSK TB set, were confirmed as GlmU interacting compounds and shown to inhibit the growth of M. tuberculosis. These results show that knowledge of the kinetic and catalytic mechanism enabled optimisation of a thorough screening approach that identified novel inhibitors that demonstrated that GlmU acetyltransferase activity is essential for M. tuberculosis growth.

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