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Structural Studies on Degradation Mechanisn of Organohalides by Dehalogenase Using Protein-Engineering Techniques

利用蛋白质工程技术进行脱卤酶降解有机卤化物机理的结构研究

基本信息

批准号：
09660088
负责人：
HATA Yasuo
金额：
$ 2.11万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Dehalogenases for decomposing harmful organohalides must be developed as soon as possible to clean up environmental pollution. In the present research, structural studies at atomic level on mechanism of degrading substrates organohalides by Pseudomonas sp. YL L-2-haloacid dehalogenase, using protein-engineering techniques. The 2 * X-ray crystal structure analysis of the native enzyme has revealed that each subunit of the dimeric enzyme consists of two domains : the core-domain having a alpha/beta structure with the active site, and four-helix bundle domain for dimerization. Subsequently, 2 * crystal structures of complexes prepared by soaking the S175A mutant crystals in solutions of various substrates, L-2-haloacids, have revealed those of ester intermediates where the side-chain carboxyle oxygen is covalently bonded to the C2 atom of the substrate. In the intermediates, the region of Asp10-Thr14 moves towards the active site, and the substrates lose the halogen and change their configuration from L- to D-forms. In each case, moreover, a new water molecule has been found in the vicinity of the Serl75 hydroxyle. In case of the substrate L-2-haloamide, the electron density peak, which can be assigned as the halide ion released from the substrate, has occurred near the side chain of Arg41 which is expected to be involved in abstraction of the halogen from the substrate. In each intermediate, the carboxyle group of the substrate has been stabilized by hydrogen bonds with the Ser118 hydroxyle and the main-chain amino group of Asn119, and the arkyl group of the substrate has been stabilized byhydrophobic interactions in the hydrophobic pocket.

为了解决环境污染问题，必须尽快开发降解有害有机卤化物的脱卤酶。本研究利用蛋白质工程技术，在原子水平上对假单胞菌YL-2-卤酸脱卤酶降解有机卤化物的机理进行了结构研究。天然酶的2 * X射线晶体结构分析表明，二聚体酶的每个亚基由两个结构域组成：具有活性位点的α/β结构的核心结构域和用于二聚化的四螺旋束结构域。随后，通过将S175 A突变体晶体浸泡在各种底物L-2-卤代酸的溶液中制备的复合物的2 * 晶体结构揭示了酯中间体的那些，其中侧链羧基氧共价键合到底物的C2原子上。在中间体中，Asp 10-Thr 14区域向活性位点移动，底物失去卤素并将其构型从L-变为D-形式。此外，在每种情况下，在Ser 175羟基附近都发现了一个新的水分子。在底物L-2-卤代酰胺的情况下，电子密度峰（其可以被认为是从底物释放的卤离子）出现在Arg 41的侧链附近，预期Arg 41参与从底物中提取卤素。在每个中间体中，底物的羧基通过与Ser 118羟基和Asn 119主链氨基的氢键而稳定，底物的芳基通过疏水口袋中的疏水相互作用而稳定。