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

• 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; β-lactam; infectious disease; inhibitor; lactamase; β-ラクタム剤; 加水分解酵素

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-内酰胺酶会催化包括碳青霉烯在内的大多数β-内酰胺抗生素的水解，目前尚无这种酶的有效抑制剂。我们的最终目标是开发基于结构的金属β-内乳糖酶抑制剂，我们设置了两个子主题：（1）从静脉质子菌中制备IMP-1金属 - β-内酰胺酶的载脂蛋白酶。可以通过应用相当高的温度（30 refe of Edta（30 r）（50 mm）（50 mm）（50 mm）（50 mm）（50 mm）（30 mm），apo-imp-1酶获得（50 mm），50 mm（30 r）（30 r）（30 r）（30 r）（ MM，pH 6.5，1.0 m NaCl含有30％甘油）。通过使用短凝胶过滤柱（PD-10）在4℃中使用短凝胶EDTA。通过原子吸收光谱法检查制备的Apo-IMP-1酶的Zn（II）含量为每一个IMP-1分子0.076，表明Apo-Imp-1酶的活性小于未经处理的EDTA的95％。在通过这种方法制备的Apo-Imp-1中，通过添加少量的Zn（NO_3）_2 _6H_2O。（2）VIM-2金属 - β-内酰胺酶的结晶和晶体学从pseudomonas eruginosa中添加了铜豆氨基甲基甲基酸酯，从而完全恢复了酶活性。发现RAC-2-苯基丙基-3-甲基丙酸苯基霉素是VIM-2的潜在抑制剂。通过X射线晶体学确定了VIM-2-苯基C3SH络合物的结构2.3 A.该结构表明，苯基克3SH的硫醇基团桥接到两个锌（II）离子和苯基基团与Tyr67（47）相互作用的tyr67（47）在活性位点附近的loop1上与π-π堆积相互作用相互作用。亚甲基与位于ch-π相互作用的Loop1底部的Phe61（42）相互作用。与天然结构（PDB代码：1KO3）相比，LOOP2上的ARG228（185）和ASN233（190）观察到动态运动。这些结果表明，上述四个保留在抑制剂或底物的结合和识别以及在VIM-2酶中稳定环的结合和识别中起重要作用。