Medical Microbiology and Molecular Biology Lab., Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India.
Medical Microbiology and Molecular Biology Lab., Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India.
Int J Biol Macromol. 2018 Jun;112:1272-1277. doi: 10.1016/j.ijbiomac.2018.02.091. Epub 2018 Feb 15.
New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1 were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution.
新德里金属β-内酰胺酶-1 是碳青霉烯酶之一，几乎能水解所有β-内酰胺类抗生素。到目前为止，已经报道了 17 种不同的 NDM 变体，它们的序列要么通过单个氨基酸取代，要么通过多个氨基酸取代而有所不同。因此，了解其结构和功能关系非常重要。在早期的研究中，已经研究了活性位点残基的作用，但没有详细研究非活性位点残基。因此，我们通过突变其一些非活性位点残基来进一步理解其结构和功能关系。通过基于 PCR 的定点诱变，在实验室中生成了 NDM-1 的突变体，将 123 位的 Q 突变为 A。与野生型相比，NDM-1 的亚胺培南和美罗培南的 MIC 增加了 2 倍，因此水解活性（Kcat/Km）增强。GOLD 适应度评分也有利于动力学数据。远紫外圆二色性（CD）测定α-螺旋含量的二级结构，显示出显著的构象变化。我们得出结论，非活性位点氨基酸残基在 NDM-1 的催化活性中起着重要作用。这项研究还提供了通过自然进化产生新变体的见解。
