Involvement of the non-active site Residues in the Catalytic Activity of NDM-4 Metallo beta-lactamase

Abstract

The rise of New Delhi metallo beta-lactamase (NDM) producing bacteria imposes a significant threat to the treatment of bacterial infections due to their broad spectrum against beta-lactams. The activity of metallo beta-lactamases is affected by active site residues as well as residues near the active site. Therefore, we aimed to identify the amino acid residues around the active site of NDM-4 which influence its function. To achieve that, seven substitution mutations (S191A, D192A, S213A, K216A, S217A, D223A and D225A) of NDM-4 were generated through site-directed mutagenesis. Out of these, expression of NDM-4_D192A and NDM-4_S217A in Escherichia coli cells increased the beta-lactam susceptibility as compared to NDM-4. Further, proteins were purified to assess the effect of substitution mutations on zinc content, in vitro catalytic efficiency, and stability of NDM-4. The catalytic efficiency was reduced for these mutants (D192A and S217A) towards beta-lactam substrates, while the thermal stability remained insubstantial as compared to NDM-4. However, the purified NDM-4_D192A exhibited altered zinc content. In silico studies reveal that these changes might be the outcomes of alterations in hydrogen bonding networks and substrate interactions. Taken together, we infer that the D192 and the S217 residues play a substantial role in the activity of NDM-4.