title: Kinetic modelling of GlmU reactions - prioritization of reaction for therapeutic application.
abstract: Mycobacterium tuberculosis(Mtu), a successful pathogen, has developed resistance against the existing anti-tubercular drugs necessitating discovery of drugs with novel action. Enzymes involved in peptidoglycan biosynthesis are attractive targets for antibacterial drug discovery. The bifunctional enzyme mycobacterial GlmU (Glucosamine 1-phosphate N-acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase) has been a target enzyme for drug discovery. Its C- and N- terminal domains catalyze acetyltransferase (rxn-1) and uridyltransferase (rxn-2) activities respectively and the final product is involved in peptidoglycan synthesis. However, the bifunctional nature of GlmU poses difficulty in deciding which function to be intervened for therapeutic advantage. Genetic analysis showed this as an essential gene but it is still unclear whether any one or both of the activities are critical for cell survival. Often enzymatic activity with suitable high-throughput assay is chosen for random screening, which may not be the appropriate biological function inhibited for maximal effect. Prediction of rate-limiting function by dynamic network analysis of reactions could be an option to identify the appropriate function. With a view to provide insights into biochemical assays with appropriate activity for inhibitor screening, kinetic modelling studies on GlmU were undertaken. Kinetic model of Mtu GlmU-catalyzed reactions was built based on the available kinetic data on Mtu and deduction from Escherichia coli data. Several model variants were constructed including coupled/decoupled, varying metabolite concentrations and presence/absence of product inhibitions. This study demonstrates that in coupled model at low metabolite concentrations, inhibition of either of the GlmU reactions cause significant decrement in the overall GlmU rate. However at higher metabolite concentrations, rxn-2 showed higher decrement. Moreover, with available intracellular concentration of the metabolites and in vivo variant of model, uncompetitive inhibition of rxn-2 caused highest decrement. Thus, at physiologically relevant metabolite concentrations, targeting uridyltranferase activity of Mtu GlmU would be a better choice for therapeutic intervention.
authors.affiliation: Innovative Medicine for Infection (iMed Infection), AstraZeneca India Pvt. Ltd., Bangalore, Karnataka, India.
authors.forename: Vivek K
authors.initials: VK
authors.lastname: Singh
authors.forename: Kaveri
authors.initials: K
authors.lastname: Das
authors.forename: Kothandaraman
authors.initials: K
authors.lastname: Seshadri
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055162:Biocatalysis; D019295:Computational Biology; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology
chemical_list: D004791:Enzyme Inhibitors; D000123:Acetyltransferases; C428383:glucosamine-1-phosphate acetyltransferase; D000117:Acetylglucosamine