Shapiro Adam B, Gao Ning, Gu Rong-Fang, Thresher Jason
Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA.
Reagents and Assay Development, Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451, USA.
Anal Biochem. 2014 Oct 15;463:15-22. doi: 10.1016/j.ab.2014.06.004. Epub 2014 Jun 16.
High-molecular-weight penicillin-binding proteins (PBPs) are essential integral membrane proteins of the bacterial cytoplasmic membrane responsible for biosynthesis of peptidoglycan. They are the targets of antibacterial β-lactam drugs, including penicillins, cephalosporins, and carbapenems. β-Lactams covalently acylate the active sites of the PBP transpeptidase domains. Because β-lactams are time-dependent inhibitors, quantitative assessment of the inhibitory activity of these compounds ideally involves measurement of their second-order acylation rate constants. We previously described a fluorescence anisotropy-based assay to measure these rate constants for soluble constructs of PBP3 (Anal. Biochem. 439 (2013) 37-43). Here we report the expression and purification of a soluble construct of Pseudomonas aeruginosa PBP2 as a fusion protein with NusA. This soluble PBP2 was used to measure second-order acylation rate constants with the fluorescence anisotropy assay. Measurements were obtained for mecillinam, which reacts specifically with PBP2, and for several carbapenems. The assay also revealed that PBP2 slowly hydrolyzed mecillinam and was used to measure the rate constant for this deacylation reaction.
高分子量青霉素结合蛋白（PBPs）是细菌细胞质膜中负责肽聚糖生物合成的必需整合膜蛋白。它们是包括青霉素、头孢菌素和碳青霉烯在内的抗菌β-内酰胺类药物的作用靶点。β-内酰胺类药物与PBP转肽酶结构域的活性位点共价酰化。由于β-内酰胺类药物是时间依赖性抑制剂，对这些化合物抑制活性的定量评估理想情况下涉及测量其二阶酰化速率常数。我们之前描述了一种基于荧光各向异性的测定方法，用于测量PBP3可溶性构建体的这些速率常数（《分析生物化学》439卷（2013年）第37 - 43页）。在此，我们报告了铜绿假单胞菌PBP2可溶性构建体作为与NusA融合蛋白的表达和纯化。这种可溶性PBP2用于通过荧光各向异性测定法测量二阶酰化速率常数。对特异性与PBP2反应的美西林以及几种碳青霉烯类药物进行了测量。该测定还表明PBP2缓慢水解美西林，并用于测量这种脱酰化反应的速率常数。
