Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Chemical Biology Innovation Laboratory, College of Chemistry and Materials Science, Northwest University, 1 Xuefu Avenue, Xi'an, 710127, PR China.
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Chemical Biology Innovation Laboratory, College of Chemistry and Materials Science, Northwest University, 1 Xuefu Avenue, Xi'an, 710127, PR China.
Anal Biochem. 2019 Aug 1;578:29-35. doi: 10.1016/j.ab.2019.05.002. Epub 2019 May 6.
The d,d-dipeptidase enzyme VanX is the main cause of vancomycin resistance in gram-positive bacteria because of hydrolysis of the D-Ala-D-Ala dipeptide used in cell-wall biosynthesis. Continuous assay of VanX has proven challenging due to lack of a chromophoric substrate. Here, we report a direct approach for continuous assay of VanX in vitro and in vivo from hydrolysis of D-Ala-D-Ala, based on the heat-rate changes measured with isothermal titration calorimetry (ITC). With the ITC approach, determination of kinetic parameters of VanX hydrolyzing D-Ala-D-Ala and the inhibition constant of d-cysteine inhibitor yielded K of 0.10 mM, k of 11.5 s, and K of 18.8 μM, which are consistent with the data from ninhydrin/Cd(II) assays. Cell-based ITC studies demonstrated that the VanX expressed in E. coli and in clinical strain VRE was inhibited by d-cysteine with IC values of 29.8 and 28.6 μM, respectively. Also, the total heat from D-Ala-D-Ala (4 mM) hydrolysis decreases strongly (in absolute value) from 1.26 mJ for VRE to 0.031 mJ for E. faecalis, which is consistent with the large MIC value of vancomycin of 512 μg/mL for VRE and the much smaller value of 4 μg/mL for E. faecalis. The ITC approach proposed here could be applied to screen and evaluate small molecule inhibitors of VanX or to identify drug resistant bacteria.
D,d-二肽酶 VanX 是革兰氏阳性菌产生万古霉素耐药性的主要原因，因为它可以水解用于细胞壁生物合成的 D-Ala-D-Ala 二肽。由于缺乏生色底物，连续检测 VanX 一直具有挑战性。在这里，我们报告了一种基于等温滴定微量热法（ITC）测量的热率变化，用于体外和体内连续检测 D-Ala-D-Ala 水解的 VanX 的直接方法。通过 ITC 方法，测定了 VanX 水解 D-Ala-D-Ala 的动力学参数和 d-半胱氨酸抑制剂的抑制常数，得到 K 的值为 0.10mM，k 的值为 11.5s，K 的值为 18.8μM，与茚三酮/Cd(II) 测定法的数据一致。基于细胞的 ITC 研究表明，在大肠杆菌和临床菌株 VRE 中表达的 VanX 被 d-半胱氨酸抑制，IC 值分别为 29.8 和 28.6μM。此外，来自 D-Ala-D-Ala（4mM）水解的总热量绝对值从 VRE 的 1.26mJ 强烈下降到 E. faecalis 的 0.031mJ，这与 VRE 的万古霉素 MIC 值为 512μg/mL 且 E. faecalis 的值小得多为 4μg/mL 一致。这里提出的 ITC 方法可用于筛选和评估 VanX 的小分子抑制剂或鉴定耐药菌。
