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<p>New Delhi Metallo-β-lactamase-1 (NDM-1) belongs to the carbapenemase family of enzymes, which can hydrolyse a wide range of β-lactam antibiotics. To date, 91 NDM-1 variants have been reported globally. In this study, we generated a novel mutant, NDM-1 D254A, through site-directed mutagenesis to investigate the functional role of non-active site residue Asp254. The wild-type NDM-1 and D254A mutant were cloned, purified, and systematically analyzed using steady-state kinetic assays and circular dichroism spectroscopy. Subsequently, we conducted Insilico studies, which include molecular docking and molecular dynamics (MD) simulations. The D254A mutant has reduced catalytic activity, thus indicating impaired β-lactam hydrolysis. Structural and computational analysis revealed that Asp254 plays an essential role in maintaining the protein stability. These findings illustrate the importance of Asp254 in maintaining the NDM-1 activity and provide the mechanistic insights into the structure-function relationship. This study enhances our understanding of NDM-1 variants and may aid the rational design of targeted inhibitors to counteract the rising global threat of NDM-1 mediated antibiotic resistance.</p>

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Structural and functional characterization of the New Delhi metallo beta-lactamase-1 (NDM-1) D254A mutant: a biochemical, microbiological and bioinformatics study

  • 期刊论文

  • 发表日期：

    2025年12月31日

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  • Abid Ali,
  • Kulsum Fatima,
  • Nabeela Farhat,
  • Asad U. Khan

摘要

New Delhi Metallo-β-lactamase-1 (NDM-1) belongs to the carbapenemase family of enzymes, which can hydrolyse a wide range of β-lactam antibiotics. To date, 91 NDM-1 variants have been reported globally. In this study, we generated a novel mutant, NDM-1 D254A, through site-directed mutagenesis to investigate the functional role of non-active site residue Asp254. The wild-type NDM-1 and D254A mutant were cloned, purified, and systematically analyzed using steady-state kinetic assays and circular dichroism spectroscopy. Subsequently, we conducted Insilico studies, which include molecular docking and molecular dynamics (MD) simulations. The D254A mutant has reduced catalytic activity, thus indicating impaired β-lactam hydrolysis. Structural and computational analysis revealed that Asp254 plays an essential role in maintaining the protein stability. These findings illustrate the importance of Asp254 in maintaining the NDM-1 activity and provide the mechanistic insights into the structure-function relationship. This study enhances our understanding of NDM-1 variants and may aid the rational design of targeted inhibitors to counteract the rising global threat of NDM-1 mediated antibiotic resistance.