1998 Fiscal Year Final Research Report Summary
Structural Studies on Degradation Mechanisn of Organohalides by Dehalogenase Using Protein-Engineering Techniques
| Project/Area Number |
09660088
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
HATA Yasuo Kyoto University, Institute for Chemical Research, Associate Professor, 化学研究所, 助教授 (10127277)
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| Project Period (FY) |
1997 – 1998
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| Keywords | dehalogenase / X-ray crystal analysis / reaction intermediate / mutant enzyme / four-dimensional Structure / dehalogenation / SN2 reaction |
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| Research Abstract |
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.
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