Studying the mechanism and action of Purine biosynthesis enzymes in Mycobacterium Tuberculosis H37RV

MtbSAICAR and MtbpurD are two essential enzymes in the Mycobacterium Tuberculosis purine biosynthesis pathway. The aim of this work is to improve our understanding of these enzymes by characterising the kinetics and determining their structures. MtbpurD has been expressed and purified but it failed to crystallise, and so homology modelling was used to compare it to homologues enzymes. MtbSAICAR was successfully crystallised and the X-ray crystal structures determined. The kinetic parameters in solution were also measured. The MtbSAICAR structures were solved in apo form and in complex with substrate and inhibitor ligands, the binding of ligands does not induce major conformation changes. Although all the solved structures are isomorphous, there are some differences in mobility seen upon ligand binding. Comparing MtbSAICAR with homologues enzymes revealed that despite variation in oligomeric organisation, they share similar folds and active sites. The similarities and differences have been evaluated and compared using both global and local alignments. Two models have been proposed for the mechanism and action of SAICARs. The structures of MtbSAICAR presented here are consistent with the mechanism in which the first step is the phosphorylation of the substrate (CAIR) by ATP, rather than the formation of a phosphoenzyme intermediate. The comparisons between MtbSAICAR and the human equivalent enzyme (HsSAICAR) show differences which might have the potential to be exploited as a target for specific inhibitors as new drugs to combat tuberculosis.