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

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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.

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Acknowledgements

The authors are highly thankful to Prof. Amitabha Bhattacharjee, Department of Microbiology, Assam University, Silchar, India for providing blaNDM−4 carrying E. coli ST44.

Funding

This work was funded partially by two grants, one from the Department of Biotechnology (DBT), Government of India [grant # BT/PR24255/NER/95/716/2017] and another from Council of Scientific and Industrial Research (CSIR), Govt. of India [grant # 27(0367)/20/EMR-II ].

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Authors and Affiliations

  1. Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, 721302, India

    Jyoti Verma

  2. Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, 721302, India

    Diamond Jain, Aditya Prasad Panda, Shri Kant, Gaurav Kumar & Anindya Sundar Ghosh

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  1. Jyoti Verma
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Contributions

Jyoti Verma: Performed experiment and wrote the first draft; Diamond Jain: Performed experiment, edited the manuscript, prepared the revised manuscript; Aditya Prasad Panda: Bioinformatics analysis and relevant writing; Shri Kant: Bioinformatics analysis and relevant writing; Gaurav Kumar: provided relevant suggestions for improvement; Anindya S Ghosh: Conceptualization, supervision and preparation of the final manuscript.

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Correspondence to Anindya Sundar Ghosh.

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Verma, J., Jain, D., Panda, A.P. et al. Involvement of the non-active site Residues in the Catalytic Activity of NDM-4 Metallo beta-lactamase. Protein J 42, 316–326 (2023). https://doi.org/10.1007/s10930-023-10124-6

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