2000 Fiscal Year Final Research Report Summary
TOXIN-DIRECTED MOLECULAR CONVERSION SYSTEM WITH YEAST HARBORING HIGHLY ACTIVATED P450 ENZYME BY ARTIFICIAL MUTAGENESIS
| Project/Area Number |
09480130
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
環境保全
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| Research Institution | Tohoku University |
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Principal Investigator |
SHIMIZU Toru TOHOKU UNIVERSITY, INSTITUTE FOR CHEMICAL REACTION SCIENCE REGISTERED NUMBER, 反応化学研究所, 教授 (40118956)
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| Co-Investigator(Kenkyū-buntansha) |
DAFF Simon TOHOKU UNIVERSITY, INSTITUTE FOR CHEMICAL REACTION SCIENCE, RESEARCH ASSOCIATE, 反応化学研究所, 助手 (50292298)
SATO Hideaki TOHOKU UNIVERSITY, INSTITUTE FOR CHEMICAL REACTION SCIENCE, RESEARCH ASSOCIATE, 反応化学研究所, 助手 (60271996)
SAGAMI Ikuko TOHOKU UNIVERSITY, INSTITUTE FOR CHEMICAL REACTION SCIENCE, LECTURER, 反応化学研究所, 講師 (10143033)
MATSUI Toshitaka TOHOKU UNIVERSITY, INSTITUTE FOR CHEMICAL REACTION SCIENCE, RESEARCH ASSOCIATE, 反応化学研究所, 助手 (90323120)
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| Project Period (FY) |
1997 – 2000
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| Keywords | HALOETHANES / HEME ENZYMES / DEHALOGENATIONS / CYTOCHROME P450 / MUTATIONS / ELECTRON TRANSFER / AZO COMPOUNDS / MOLECULAR SWITCH |
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| Research Abstract |
(1) High utility of Saccharomyces cerevisiae harboring rat liver P450 cDNA in haloethanes dehalogenations : Liver P450 is monooxygenase and uses O_2 and electrons from NADPH.Liver P450s have thousands of toxic organic substrates and work as detoxic enzyme to help chemicals to be eliminated from the body. Yeast harboring rat liver P450 1A2 efficiently degraded trichloroethylene, pentachloroethane and hexachloroethane. Mutations on the substrate binding surface and heme distal site enormously enhanced the catalytic activity toward those haloethanes. Since liver P450 catalyze degradation of thousands of chemicals, this method is promising for chemical-directed degradation of environmental pollutants.
(2) Molecular switch of NO synthase oxygenase domain-P450BM3 reductase domain chimeric enzyme : NO synthase is composed of an oxygenase domain with P450-like heme active site and a reductase domain which is similar to NADPH-P450 reductase and the NO formation activity and interdomain electron
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transfer is controlled by Ca^<2+>/calmodulin. P450BM3 is also composed of the P450 oxygenase domain and the reductase domain but the catalysis and the electron transfer are not controlled by Ca^<2+>/calmodulin. In order to construct a novel enzyme with molecular switch system, we generated a chimeric protein composed of the NO synthase oxygenase domain and the P450BM3 reductase domain. The new chimeric enzyme functions including NO formation activity, substrate binding and electron transfer were controlled by Ca^<2+>/calmodulin. Thus, this protein eaxgineering approach sheds light for application of the enzymatic system with molecular switch to environmental degradation.
(3) Azo reduction of neuronal nitric oxide synthase :
Nitric oxide synthase catalyzes NO formation from L-Arg. This enzyme efficiently catalyzed the decomposition of one of azo compounds, which are often environmental pollutamts and carcinogen. This decomposition was controlled with Ca^<2+>/calmodulin as a molecular switch. Therefore, we found that NO synthase could work to degrade environmental chemical under specific conditions. Less
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[Publications] Toru Shimizu, Ikuko Sagami, Simon Daff, Tomomasa Shimanuki, Shigeyoshi Fujiwara, Steve Chapman, and Andrew Munro: "Electron Transfer Modulation of the P450 System"The 11th International Conference on Cytochrome P450, August 29-September 2, 1999, Sendai, Japan..
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「研究成果報告書概要(欧文)」より
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[Publications] Toru Shimizu, Ikuko Sagami, Simon Daff, Tomomasa Shimanuki, Shigeyoshi Fujiwara, Andrew Munro, and Steve Chapman: "Interdomain Electron Transfer of Fusion Proteins Composed of Oxygenase Domain and Reductase Domain : Cytochrome P450BM3 and Nitric Oxide Synthase"2nd BRAIN INTERNATIONAL SEMINAR-Cytochrome P450 and Plant Genetic Engineering, November 24-25, 1999, Kobe, Japan..
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[Publications] Michihito Miyajima, Makoto Umeno, Ikuko Sagami, Simon Daff, Catharina T.Migita, Masao Ikeda-Saito, and Toru Shimizu: "Azo Bond Reduction of Methyl Red with Nitric Oxide Synthase"The First International Conference-Biology, Chemistry and Therapeutic Applications of Nitric Oxide, June 3-7, 2000, San Francisco, U.S.A..
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「研究成果報告書概要(欧文)」より
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