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-内酰胺酶催化包括碳青霉烯类在内的大多数β-内酰胺类抗生素的水解，目前还没有有效的酶抑制剂。我们的最终目标是开发基于结构的金属-β-内酰胺酶抑制剂，我们设定了两个主题：(1)粘质沙雷氏菌金属-β-内酰胺酶的制备：在较高的温度(30℃)、高浓度的乙二胺四乙酸乙二酯(50 MM)和pH 6.5的介质(MOPS-NaOH，50 mM，pH 6.5，含30%甘油的1.0M氯化钠)中，用较高的温度(30℃)，高浓度的乙二胺四乙酸乙二酯(50 MM)可以获得脱氧酶。原子吸收光谱分析表明，制备的apo-IMP-1酶的锌含量为0.076个/imp-1分子，活性低于未经处理的EDTA的95%。在用这种方法制备的apo-β-1中，加入少量过量的锌(NO_3)_2·6H_2O可以很好地恢复酶的活性。(2)铜绿假单胞菌VIM-2金属-IMP-内酰胺酶与巯基羧酸盐抑制剂复配的结晶和结晶学最近，我们发现Rac-2-苯丙基-3-巯基丙酸苯基C3SH是VIM-2的一种有效的抑制剂。用X-射线单晶衍射法测得VIM-2-苯基C3SH络合物的结构为2.3A。结构表明，苯基C3SH的硫醇通过π-π堆积作用与活性中心附近的环1上的酪氨酸67(47)发生相互作用。亚甲基通过CH-π相互作用与位于loop1底部的Phe61(42)相互作用。与天然结构(PDB代码：1K03)相比，在loop2上观察到Arg228(185)和Asn233(190)中的动态运动。这些结果表明，上述四个残基在抑制物或底物的结合和识别以及稳定VIM-2酶的环路中起着重要作用。