权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Structure-based Design of Selective metallo-β-lactamase

基于结构的选择性金属-β-内酰胺酶设计

基本信息

批准号：
18390038
负责人：
KUROSAKI Hiromasa
金额：
$ 10.63万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390038/
关键词：
β-lactam infectious disease inhibitor lactamase β-ラクタム剤加水分解酵素

项目摘要

Metallo-β-lactamases catalyzes the hydrolysis of most β-lactam antibiotics including carbapenems, and there are currently no potent inhibitors of such enzymes. Our ultimate goal is to develop structure-based inhibitors of metallo-β-lactamases and we set two subthemes :(1)Preparation of apoenzyme of IMP-1 metallo-β-lactamase from Seratia marcescens.The apo-IMP-1 enzyme can be obtained by application of rather high temperature(30℃),high concentration of EDTA(50 mM)and the medium of pH 6.5(MOPS-NaOH, 50 mM, pH 6.5, 1.0 M NaCl containing 30% glycerol).Excess EDTA was removed by use of short gel filtration column (PD-10)at 4℃. The Zn(II)content of the prepared apo-IMP-1 enzyme was checked by atomic absorption spectrometry to be 0.076 per an IMP-1 molecule, indicating that the activity of apo-IMP-1 enzyme is less than 95 % of the untreated EDTA. In apo-IMP-1 prepared by this method, the enzymatic activity was perfectly recovered by the addition of a small excess of Zn(NO_3)_2・6H_2O.(2) Crystallization and crystallographic of VIM-2 metallo-β-lactamase from Pseudomonas aeruginosa Complexed with a Mercaptocarboxylate InhibitorRecently, we found rac-2-Phenylpropyl-3-mercaptopropionic acid, PhenylC3SH, was found to be a potent inhibitor of VIM-2. The structure of the VIM-2-PhenylC3SH complex was determined by X-ray crystallography to 2.3 A. The structure revealed that the thiol group of PhenylC3SH bridged to the two zinc (II) ions and the phenyl group interacted with Tyr67 (47) on loop1 near the active site, by π-π stacking interactions. The methylene group interacted with Phe61 (42) located at the bottom of loop1 though CH-π interactions. Dynamic movements were observed in Arg228 (185) and Asn233 (190) on loop2, compared with the native structure (PDB code: 1KO3). These results suggest that the above-mentioned four residues play important roles in the binding and recognition of inhibitors or substrates and in stabilizing a loop in the VIM-2 enzyme.

金属-β-内酰胺酶催化大多数β-内酰胺抗生素（包括碳青霉烯类）的水解，目前还没有此类酶的有效抑制剂。（1）从粘质沙雷氏菌（Seratiamarcescens）中制备IMP-1金属β-内酰胺酶的脱辅基酶（apo-IMP-1 enzyme），采用高温（30℃）、高浓度EDTA（50 mM）、pH6.5（MOPS-NaOH，50 mM，pH6.5，1.0 M NaCl，含30%甘油）的条件，在4℃下用短凝胶过滤柱（PD-10）除去过量的EDTA，得到apo-IMP-1酶。通过原子吸收光谱法检查制备的apo-IMP-1酶的Zn（II）含量为0.076/IMP-1分子，表明apo-IMP-1酶的活性小于未处理EDTA的95%。在此方法制备的apo-IMP-1中，加入少量过量的Zn（NO_3）_2·6H_2O即可完全恢复酶活性。(2)铜绿假单胞菌Vim-2金属β-内酰胺酶与巯基羧酸抑制剂复合物的结晶和晶体学研究Vim-2-PhenylC 3SH复合物的结构通过X射线晶体学测定为2.3 A。结构分析表明，PhenylC 3SH的巯基与两个Zn（II）离子桥连，苯基与活性中心附近环1上的Tyr 67（47）通过π-π堆积作用相互作用。亚甲基通过CH-π相互作用与位于环1底部的Phe 61（42）相互作用。与天然结构（PDB代码：1 KO 3）相比，在环2上的Arg 228（185）和Asn 233（190）中观察到动态运动。这些结果表明，上述四个残基在结合和识别抑制剂或底物以及稳定Vim-2酶中的环中起重要作用。