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2000 Fiscal Year Final Research Report Summary

TOXIN-DIRECTED MOLECULAR CONVERSION SYSTEM WITH YEAST HARBORING HIGHLY ACTIVATED P450 ENZYME BY ARTIFICIAL MUTAGENESIS

Research Project

Project/Area Number 09480130
Research Category

Grant-in-Aid for Scientific Research (B).

Allocation TypeSingle-year Grants
Section一般
Research Field 環境保全
Research InstitutionTohoku University

Principal Investigator

SHIMIZU Toru  TOHOKU UNIVERSITY, INSTITUTE FOR CHEMICAL REACTION SCIENCE REGISTERED NUMBER, 反応化学研究所, 教授 (40118956)

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)
Project Period (FY) 1997 – 2000
KeywordsHALOETHANES / HEME ENZYMES / DEHALOGENATIONS / CYTOCHROME P450 / MUTATIONS / ELECTRON TRANSFER / AZO COMPOUNDS / MOLECULAR SWITCH
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 … More 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

  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] T.Shimizu,I.Sagami,: "Azo Reduction of Methyl Red by Neuronal Nitric Oxide Synthase : The Important Role of FMN in Catalysis"Biochem.Biophys.Res.Commun.. Vol.275. 752-758 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Shimizu,I.Sagami: "Azo Bond Reduction of Methyl Red with Nitric Oxide Synthase"Nitric Oxide, Biology and Chemistry. Vol.4. 212 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Shimizu,I.Sagami,: "Control of Electron Transfer in Neuronal NO Synthase"Biochem.Soc.Trans.. (印刷中). (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Shimizu,I.Sagami: "The Electron Transfer Reaction of Niric-oxide Synthase"Coord.Chem.Rev.. 189(印刷中). (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kazutaka Yanagita, Ikuko Sagami, and Toru Shimizu: "High Utility of Saccharomyces cerevisiae Harboring Rat Liver Cytochrome P450 1A2 cDNA in Haloethanes Dehalogenations"Chemistry Lett.. 561-562 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Toru Shimizu: "Diverse Role of Conserved Aromatic Amino Acids in the Electron Transfer of Cytochrome P450 Catalytic Functions : Site-directed Mutagenesis Studies"Recent Res.Devel. in Pure & Applied Chem.. 1. 169-175 (1997)

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      「研究成果報告書概要(欧文)」より
  • [Publications] Kazutaka Yanagita, Ikuko Sagami, and Toru Shimizu: "Distal Site and Surface Mutations of Cytochrome P450 1A2 Markedly Enhance Dehalogenation of Chlorinated Hydrocarbons"Arch.Biochem.Biophys.. 346. 269-276 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kazutaka Yanagita, Ikuko Sagami, Simon Daff, and Toru Shimizu: "Marked Enhancement in the Reductive Dehalogenation of Hexachloroethane by a Thr319Ala Mutation of Cytochrome P450 1A2"Biochem.Biophys.Res.Commun.. 249. 678-682 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Michihito Miyajima, Ikuko Sagami, Simon Daff, Catharina T.Migita, and Toru Shimizu: "Azo Reduction of Methyl Red by Neuronal Nitric Oxide Synthase : The Important Role of FMN in Catalysis"Biochem.Biophys.Res.Commun.. 275. 752-758 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] S.Daff, M.A.Noble, D.H.Craig, S.L.Rivers, S.K.Chapman, A.W.Munro, S.Fujiwara, E.Rozhkova, I.Sagami, and T.Shimizu": "Control of Electron Transfer in Neuronal NO Synthase"Biochem.Soc.Trans.. (in press). (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ikuko Sagami, Yuko Sato, Tomoko Noguchi, Michihito Miyajima, Elena Rozhkova, Simon Daff, and Toru Shimizu: "The Electron Transfer Reaction of Nitric-oxide Synthase"Coord.Chem.Rev. (in press). (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kazutaka Yanagita, Ikuko Sagami, and Toru Shimizu: "Application of P450 Heterologus Expression Systems for Environmental Biodegradation"BRAIN INTERNATIONAL SEMINAR-Cytochrome P450 and Plant Genetic Engineering, October 30-31,1997 Tsukuba, Japan..

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      「研究成果報告書概要(欧文)」より
  • [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
  • [Publications] Shigeyoshi Fujiwara, Ikuko Sagami, Simon Daff, Andy Munro, Steve Chapman, and Toru Shimizu: "Electron Transfer in the P450BM3-Nitric Oxide Synthase Chimeric Enzyme"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
  • [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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      「研究成果報告書概要(欧文)」より
  • [Publications] Shigeyoshi Fujiwara, Ikuko Sagami, Simon Daff, Andrew Munro, Steve Chapman, and Toru Shimizu: "Electron Transfer in a Chimeric Protein Composed of Cytochrome P450BM3 Oxygenase Domain and Neuronal Nitric Oxide Syntase Reductase Domain"2nd BRAIN INTERNATIONAL SEMINAR-Cytochrome P450 and Plant Genetic Engineering, November 24-25, 1999, Kobe, Japan..

    • Description
      「研究成果報告書概要(欧文)」より
  • [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..

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kazutaka, Yaganita, and Toru Shimzu: "Application of Heterologous Cytochrome P450/Yeast Expression System to Degradation of Environmental Chemical Pollutants"Tohoku University Gene Research Center Communication. vol.37. 5-6 (1997)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2002-03-26   Modified: 2021-04-07  

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