E152A substitution drastically affects NDM-5 activity.

New Delhi Metallo beta-lactamase (NDM) is of significant public health concern due to its enormous potential to hydrolyse all major beta-lactams including carbapenems. Amino acid substitutions outside the active site reportedly affect NDM beta-lactamase activities. Here, the effect of amino acid substitutions in the possible omega-like loop region of NDM-5 has been elucidated. Overall, three substitution mutations near active site of NDM-5 were done, namely, E152A, S191A and D223A and subsequently, the change in antimicrobial resistance was monitored upon expressing each mutant in a suitable host. Among the three mutants, E152A substitution on a loop near the active site resulted in significant reduction in beta-lactam antibiotic resistance as compared to NDM-5 that compelled us to conduct further studies on the E152A-substituted NDM-5. The purified NDM-5 was able to hydrolyse all the beta-lactams tested whereas the E152A mutation suppressed its activities. NDM-5 showed maximum kcat/Km ratio against penicillins and carbapenems and had lower Km as compared to NDM-5_E152A. Though, the amino acid substitution did not affect the overall folding pattern of NDM-5, significant differences in thermal stability between the wild-type and mutated protein were observed. Therefore, we infer that the E152 residue is important in regulating the beta-lactam hydrolysing properties of NDM-5.