Comparison of Verona Integron-Borne Metallo-β-Lactamase (VIM) Variants Reveals Differences in Stability and Inhibition Profiles.
Comparison of Verona Integron-Borne Metallo-β-Lactamase (VIM) Variants Reveals Differences in Stability and Inhibition Profiles.
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DOI：
10.1128/aac.01768-15
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发表时间：
2015-12-14
影响因子：
4.9
通讯作者：
Schofield CJ
中科院分区：
文献类型：
--
作者：
Makena A;Düzgün AÖ;Brem J;McDonough MA;Rydzik AM;Abboud MI;Saral A;Çiçek AÇ;Sandalli C;Schofield CJ
金属-β-内酰胺酶 (MBL) 的临床意义越来越大；临床上有用的 MBL 抑制剂的开发受到变异 MBL 快速进化的挑战。维罗纳整合子携带的金属-β-内酰胺酶 (VIM) 是分布最广泛的 MBL 酶之一，已报道了超过 40 个 VIM 变体。我们报告了 VIM-5 的晶体学分析以及 VIM-1、VIM-2、VIM-4、VIM-5 和 VIM-38 的生化和生物物理特性的比较。生产并纯化了重组 VIM 变体，并通过圆二色性分析研究了它们的二级结构和热稳定性。对一组代表性 β-内酰胺底物进行稳态动力学分析，以比较 VIM 变体的催化效率。此外，还筛选了一组金属酶抑制剂，以比较它们对不同 VIM 变体的影响。结果表明，VIM 变体的动力学参数仅有微小变化，但其热稳定性和抑制特性存在显着差异。总体而言，这些结果支持蛋白质稳定性可能是 MBL 进化的一个因素的观点，并强调了在抑制剂开发计划中筛选 MBL 变体的重要性。
Metallo-β-lactamases (MBLs) are of increasing clinical significance; the development of clinically useful MBL inhibitors is challenged by the rapid evolution of variant MBLs. The Verona integron-borne metallo-β-lactamase (VIM) enzymes are among the most widely distributed MBLs, with >40 VIM variants having been reported. We report on the crystallographic analysis of VIM-5 and comparison of biochemical and biophysical properties of VIM-1, VIM-2, VIM-4, VIM-5, and VIM-38. Recombinant VIM variants were produced and purified, and their secondary structure and thermal stabilities were investigated by circular dichroism analyses. Steady-state kinetic analyses with a representative panel of β-lactam substrates were carried out to compare the catalytic efficiencies of the VIM variants. Furthermore, a set of metalloenzyme inhibitors were screened to compare their effects on the different VIM variants. The results reveal only small variations in the kinetic parameters of the VIM variants but substantial differences in their thermal stabilities and inhibition profiles. Overall, these results support the proposal that protein stability may be a factor in MBL evolution and highlight the importance of screening MBL variants during inhibitor development programs.