Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Agricultural Bio-pharmaceutical Laboratory, College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao, China.
J Antimicrob Chemother. 2019 Apr 1;74(4):885-893. doi: 10.1093/jac/dky536.
Since the identification of VIM-1, point substitutions resulting in variants with differing hydrolytic activities have occurred, driving the evolution of the VIM enzymes. We previously detected a novel variant, VIM-48, containing 11 successive amino acid (aa) alterations in the C-terminal region compared with VIM-2. Single aa substitutions significantly change enzyme properties, but the effects of successive aa alterations have not previously been studied. Herein, we aimed to investigate the sequence and biochemical characteristics of VIM-48, including the role of the 11 successive aa substitutions.
VIM-48, VIM-2 and a truncated VIM-D(Δ) mutant missing 11 aa at the C-terminus relative to VIM-48 were characterized by antimicrobial susceptibility testing, protein expression and purification, determination of kinetic parameters, and homology modelling. Protein secondary structure and thermal stability measurements were also performed using circular dichroism spectral analysis.
Compared with blaVIM-2, blaVIM-48 conferred higher resistance to carbapenems. VIM expression in Pseudomonas putida resulted in higher MICs than in E. coli. VIM-48 demonstrated increased hydrolytic activity against carbapenems relative to VIM-2, while VIM-D(Δ) had significantly decreased catalytic efficiency compared with VIM-2 and VIM-48 as a result of aa deletion. In addition, secondary structure analysis revealed that VIM-48 had the greatest proportion of α-helices among the tested enzymes, corresponding to increased thermostability, while VIM-D(Δ) had the lowest proportion of α-helices and decreased thermostability.
VIM-48 has increased enzymatic activity and thermostability and increases host β-lactam resistance. Observed changes in the secondary structure of VIM-48 resulted from successive aa alterations. Therefore, VIM evolution likely occurs via both single and successive aa substitutions.
自从 VIM-1 的鉴定以来，导致水解活性不同的变体的点取代已经发生，推动了 VIM 酶的进化。我们之前检测到一种新型变体 VIM-48，与 VIM-2 相比，其 C 末端区域含有 11 个连续的氨基酸（aa）改变。单个 aa 取代会显著改变酶的性质，但连续 aa 改变的影响以前尚未研究过。在此，我们旨在研究 VIM-48 的序列和生化特性，包括 11 个连续 aa 取代的作用。
通过抗菌药物敏感性试验、蛋白表达和纯化、动力学参数测定以及同源建模对 VIM-48、VIM-2 和 VIM-D（Δ）突变体（相对于 VIM-48 缺失 C 末端 11 个 aa）进行了表征。还使用圆二色光谱分析进行了蛋白质二级结构和热稳定性测量。
与 blaVIM-2 相比，blaVIM-48 对碳青霉烯类药物的耐药性更高。在铜绿假单胞菌中表达 VIM 导致的 MIC 高于在大肠杆菌中表达的 MIC。与 VIM-2 相比，VIM-48 对碳青霉烯类药物的水解活性增加，而 VIM-D（Δ）由于 aa 缺失，其催化效率明显低于 VIM-2 和 VIM-48。此外，二级结构分析表明，与测试酶相比，VIM-48 具有最高比例的 α-螺旋，对应于增加的热稳定性，而 VIM-D（Δ）具有最低比例的 α-螺旋和降低的热稳定性。
VIM-48 具有增加的酶活性和热稳定性，并增加了宿主β-内酰胺类药物的耐药性。观察到 VIM-48 二级结构的变化是由于连续的 aa 取代所致。因此，VIM 的进化可能既通过单个 aa 取代又通过连续 aa 取代发生。
