Glutamic acid at position 152 and serine at position 191 are key residues required for the metallo-β-lactamase activity of NDM-7.

New Delhi metallo-β-lactamase (NDM) is of significant public-health concern due to its enormous potential to hydrolyse all of the major β-lactams, including carbapenems. Previous reports indicate that amino acid substitutions affect NDM activity despite being located outside of the active site. In this study, we attempted to identify specific mutations in loops near the active site that can influence the function of NDM-7. Overall, six substitutions were performed through site-directed mutagenesis near the active-site of NDM-7 and the change in antimicrobial resistance was subsequently monitored by expressing each mutant in a suitable bacterial host. Among the six mutants, serine at position 191 (S191) and glutamic acid at position 152 (E152) were identified as the most influencing residues for NDM-7. β-Lactam resistance of NDM-7 was remarkably affected by substitution of both residues with alanine, and the results were in accordance with the changes in kinetic parameters. Purified NDM-7 ordinarily hydrolyses β-lactams efficiently, but purified NDM-7_E152A, NDM-7_S191A and the double mutant NDM-7_E152A+S191A had lost their ability to hydrolyse the β-lactams tested, especially penicillins and carbapenems. Although the substitutions did not affect the overall folding pattern of the NDM-7 enzyme, substantial differences in thermal stability were observed. Therefore, we hypothesise that the residues S191 and E152 together play a crucial role in conferring the β-lactamase character of NDM-7.