Abstract
Glutamate racemase (MurI) is a favored target for antimicrobial development, and is an essential enzyme in M. tuberculosis, a pathogenic bacterium for which new medications are urgently needed. This thesis aims to characterise MurI from M. tuberculosis
(MurIMtb) and its close homolog from M. smegmatis (MurIMsm), and to provide biochemical and structural information for future structure-based drug discovery efforts against tuberculosis.
MurIMtb and MurIMsm were recombinantly expressed, purified, and crystallised. The crystal structures of MurIMtb and MurIMsm in complex with D-glutamate were determined to resolutions of 2.3 and 1.8 Å, respectively. The two structures are almost identical, consisting of two compact domains that adopt the canonical MurI fold, and display a complex network of substrate binding via electrostatic interactions. Structural analysis of their active sites in comparison to that of other glutamate racemase structures reveals a notable feature in mycobacterial MurI active site architecture, and
based on this feature, a possible drug design targeting the MurIMtb active site has been suggested. Moreover, a new type of MurI dimer arrangement with extensive dimer-interface interactions was observed in both mycobacterial MurI structures, termed a "side-to-side" arrangement, which is different from previously described dimer geometries for glutamate racemases. The dimeric form of mycobacterial MurI in solution was verified using analytical size-exclusion chromatography and analytical ultracentrifugation, and sedimentation equilibrium analysis of MurIMsm indicated that the enzyme forms a tight dimer with an expected Kd in the nanomolar range or lower.
Although MurIMtb and MurIMsm were shown to bind specifically to the substrates, D- and L-glutamate, neither enzyme exhibited glutamate racemase activity using two different assay methods. Single (D26R), double (D26R/R105A), and triple (D26R/Q27A/R105A) dimer-interface mutation(s) were introduced in MurIMsm to disrupt interactions at the dimeric interface, and these mutant enzymes showed glutamate racemase activity
not observed in wild-type MurIMsm. Using sedimentation velocity analysis, unlike strictly dimeric wild-type MurIMsm, a monomeric species was observed in the triple mutant at the range of concentrations at which enzyme assays were conducted, suggesting that monomeric species of MurIMsm is likely responsible for racemisation activity found in dimer-interface mutants.
The structures of MurIMtb and MurIMsm obtained here will be useful for future structure-based drug design efforts. The active dimer-interface mutants could be utilised for inhibitor screening. In addition, the finding that wild-type mycobacterial MurIs in dimeric form are inactive suggests that the dimerisation might be an additional target for drug design in an attempt to lock the monomers together.