Elucidation of the reaction mechanism of a PCB-degrading enzyme BphyC based on three-dimensional structural information.

基于三维结构信息阐明PCB降解酶BphyC的反应机制。

• 批准号: 07458251
• 负责人: MITSUI Yukio
• 金额: $ 1.15万
• 依托单位: Nagaoka University of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458251/
• 关键词: PCB; PCB-degrading enzyme; oxygenase; dioxygenase; catechol ring cleavage; X-ray analysis; protein crystallography; oligomeric enzyme.

The so-called BphC enzyme is a dioxygenase which cleaves the catechol ring moiety situated in biphenyl and its derivatives. The cleaving point is located adjacent to the two neighboring hydroxyl groups in a catechol moiety, thus the enzyme works in an extradiol fashion. This kind of enzyme performs such reactions making use of molecular oxygen and incorporates both the atomic oxygen atoms under the catalytic influence of Fe (2+) or ferrous ion.The present investigators solved the three-dimensional structure of a BphC enzyme from Pseudomonas sp. stran KKS102 by X-ray structure analysis (J.Mol. Biol. 255,735-752 (1996)). The structural information coupled with various enzyme kinetic data on wild and mutant forms of the enzyme revealed the following points.As for the catalytic mechanism :1) The site of ring cleavage appears to be specifically determined by the fact that the molecular oxygen binding site is fixed relative to the poisition and orientation of the bound substrate molecules.2) One of the conserved residues, His 194, plays a role of ctalytic base abstracting a hydrogen from the susceptible hydroxyl group.As for the substrate specificity :1) The substrate binding site is essentially hydrophobic exhibiting very high complimentarity to the catechool ring moiety of the substrates.2) The Fe ion situated in the active site is not necessarily essential for substrate binding (but essential for catalysis, of course).

所谓的BphC酶是双加氧酶，其裂解位于联苯及其衍生物中的儿茶酚环部分。裂解点位于邻苯二酚部分中的两个相邻羟基附近，因此该酶以二醇外的方式工作。本研究采用X射线结构分析（J.Mol.Chem.2003）方法，对假单胞菌（Pseudomonassp.stranKKS102）的BphC酶进行了三维结构解析，并对该酶的结构进行了表征。255，735 -752（1996））。对该酶的结构信息和各种酶动力学数据的分析表明，该酶的催化机理是：1）开环位点的特异性取决于氧结合位点相对于底物分子的位置和方向的固定性; 2）保守残基之一His 194起着从敏感羟基上夺取氢的催化碱基的作用; 1）底物结合位点基本上是疏水性的，与底物的儿茶酚环部分表现出非常高的互补性。位于活性部位的Fe离子对于底物结合不一定是必需的（当然，对于催化是必需的）。

项目成果

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