Role of conserved residues in catalytic activity of NDM-1: an approach of site directed mutagenesis and molecular dynamics

Spread of New Delhi Metallo Beta-Lactamase-1 (NDM-1) producers is a matter of major public health concerns due to drug resistance against carbapenems. The infection caused by carbapenem-resistant enterobacteria (CRE) is being classified as serious problem. To understand structural and function of NDM-1, amino acid replacement approach is considered as one of the methods to get structural insight. Therefore, we have generated novel mutation (N193A, S217A, G219A and T262A) near active site and omega-like loop to know the role of conserved residues of NDM-1. The change in antimicrobial resistance was observed upon expressing all mutant in suitable host. The minimum inhibitory concentration (MIC) of ampicillin, imipenem, meropenem, cefotaxime, cefoxitin and ceftazidime in all mutants as well as wild type NDM-1 producing strain were found reduced by 2 to 6 fold. The hydrolytic activity (kcat/km) was also reduced in all mutants as compared to wild type NDM-1. MD simulation and docking result indicated that all protein models along with wild type, showed stable and consistent behavior of RMSD, RMSF and Rg. The secondary structure of all mutant proteins showed a significant change as compared to wild type, determined by molecular dynamics simulation and CD spectrophotometer. Hence, it reveals an imperative role of near active site residues in the enzyme catalytic activity of NDM-1.