Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18
Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18
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DOI：
10.1128/aac.00985-16
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发表时间：
2016-09-01
影响因子：
4.9
通讯作者：
Shimizu-Ibuka, Akiko
中科院分区：
文献类型：
--
作者：
Furuyama, Takamitsu;Nonomura, Haruka;Shimizu-Ibuka, Akiko
IMP型金属β-内酰胺酶（MBL）是一种外源性锌金属酶，可水解多种β-内酰胺类药物，包括碳青霉烯类。在这里，我们报告的晶体结构IMP-18，从铜绿假单胞菌的MBL克隆，在2.0埃的分辨率。IMP-18的整体结构类似于IMP-1，具有α β/β α“折叠三明治”构型，但覆盖活性位点的环具有不同的构象。通过将IMP-18中可能影响环构象的氨基酸残基（Lys 44、Thr 50和Ile 69）替换为占据已充分描述的酶IMP-1中相应位置的氨基酸残基，研究了IMP-18的环构象与其动力学性质之间的关系。用Pro取代Thr 50显著改变了IMP-18的动力学性质，特别是与美罗培南有关的动力学性质，其中k（cat）/K-m值增加了一个数量级。结果表明，这是决定IMP型β-内酰胺酶动力学特性的关键残基。
IMP-type metallo-beta-lactamases (MBLs) are exogenous zinc metalloenzymes that hydrolyze a broad range of beta-lactams, including carbapenems. Here we report the crystal structure of IMP-18, an MBL cloned from Pseudomonas aeruginosa, at 2.0-angstrom resolution. The overall structure of IMP-18 resembles that of IMP-1, with an alpha beta/beta alpha "folded sandwich" configuration, but the loop that covers the active site has a distinct conformation. The relationship between IMP-18' s loop conformation and its kinetic properties was investigated by replacing the amino acid residues that can affect the loop conformation (Lys44, Thr50, and Ile69) in IMP-18 with those occupying the corresponding positions in the well-described enzyme IMP-1. The replacement of Thr50 with Pro considerably modified IMP-18' s kinetic properties, specifically those pertaining to meropenem, with the k(cat)/K-m value increased by an order of magnitude. The results indicate that this is a key residue that defines the kinetic properties of IMP-type beta-lactamases.