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Peroxygenase P450 as a Novel Bio-material

Research Project

Project/Area Number 14580616
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Bioorganic chemistry
Research InstitutionRIKEN (2003-2004)
Osaka City University (2002)

Principal Investigator

SHIRO Yoshitsugu (2003-2004)  RIKEN, Biometal Science Lab., Chief Scientist, 城生体金属科学研究室, 主任研究員 (70183051)

松永 勇 (2002)  大阪市立大学, 大学院・医学研究科, 講師 (00254425)

Co-Investigator(Kenkyū-buntansha) SUGIMOTO Hiroshi  RIKEN, Biometal Science Lab., Research Scientist, 城生体金属科学研究室, 研究員 (90344043)
KIM Misa  RIKEN, Biometal Science Lab., 城生体金属科学研究室, 基礎科学特別研究員 (40373295)
城 宣嗣  理化学研究所, 生体物理化学研究室, 主任研究員 (70183051)
小倉 壽  大阪市立大学, 大学院・医学研究科, 教授 (10115222)
Project Period (FY) 2002 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
KeywordsCytochrome P450 / Hydroxylation of Fatty Acids / Hydrogen Peroxide / Heme-enzymes / Oxygenation / X-ray Crystallography / シトクロムP450 / ペルオキシゲナーゼ / ペルオキシダーゼ / モノオキシゲナーゼ / 反応機構 / 脂肪酸
Research Abstract

Our research aim in this project was to understand the relationship of function and structure of cytochrome P450Bsβ from Bacillus subtilis, which can catalyze the hydroxylation of α- and β-positions of fatty acids using hydrogen peroxide H_2O_2 as an oxygen donor. We succeeded in making the single crystal and afterward in determining the structure (2.1Å resolution) of the ferric (Fe^<3+>) enzyme in the presence of a substrate (palimitic acid). On the basis of the structure, we prepared some mutants, and measured their enzymatic activities and spectral properties, to reveal roles of the mutated residues in the enzymatic functions (specificities in the substrate recognition, the catalytic reactions, the reaction sites, etc.). Then we proposed the molecular mechanism of the reaction catalyzed by P450Bsβ. In the mechanism, Arg242 binds with the carboxylate of the substrate fatty acid, which acts as a general acid-base catalyst in the peroxide O-O bond cleavage. The CO complex of the substrate-bound enzyme was determined. It was found that Phe79 was moved, but others were not upon the CO binding to the ferrous iron. To obtain a crystal of this enzyme in the substrate-free form, we examined the purification procedures for removal of the bound substrate by gel-filteration or by the product formation with H_2O_2. But we have not yet obtained the crystal of the substrate-free enzyme. In pursuing the structural determination of the short-lived reaction intermediate (possibly the Fe^<5+>=O state), we tried to stabilize the oxy complex of the enzyme in solution state, and then to inject two electrons with the X-ray reduction technique. We found the partial formation of the oxy complex. Finally, we discussed details of the reaction mechanism of heme-containing enzymes which utilize hydrogen peroxide.

Report

(4 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • Research Products

    (28 results)

All 2004 2003 2002 Other

All Journal Article (21 results) Publications (7 results)

  • [Journal Article] ADP Reduces the Oxygen Binding Affinity of A Sensory Histidine Kinase, FixL : Possibility of the Enhanced Reciprocating Kinase Reaction2004

    • Author(s)
      H.Nakamura, et al.
    • Journal Title

      Proc.Natl.Acad.Sci.USA 101

      Pages: 2742-2746

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Peroxide Utilizing Biocatalysts : Structural and Functional Diversity of Heme-Containing Enzymes2004

    • Author(s)
      I.Matsunaga, Y.Shiro
    • Journal Title

      Current Opinion Chem.Biol. 8

      Pages: 127-132

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Structural Basis of Human Cytoglobin for Ligand Binding2004

    • Author(s)
      H.Sugimoto, et al.
    • Journal Title

      J.Mol.Biol. 339

      Pages: 873-885

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] NO Reduction by Nitric Oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa : Characterization of Reaction Intermediates That Appear in the Single-Turnover Cycle2004

    • Author(s)
      H.Kumita, et al.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 55247-55254

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Design and Synthesis of de Novo Cytochrome c2004

    • Author(s)
      M.Ishida, et al.
    • Journal Title

      Biochemistry 43

      Pages: 9823-9833

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] D88A Mutant of Cytochrome P450nor Provides Kinetic Evidence for Direct Complex Formation with Electron Donor NADH2004

    • Author(s)
      M.Umemura, et al.
    • Journal Title

      Eur.J.Biochem. 271

      Pages: 2887-2894

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] NO Reduction by Nitric Oxide Reductase from Denitrifying Bacterium Psendomonas aeruginosa : Characterization of Reaction Intermediates That Appear in the Single-Turnover2004

    • Author(s)
      H.Kumita, et al.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 55247-55254

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Peroxide Utilizing Biocatalysis : Structural and Functional Diversity of Heme-Containing Enzymes2004

    • Author(s)
      I.Matsunaga, Y.Shiro
    • Journal Title

      Current Opinion Chem.Biol. 8

      Pages: 127-132

    • Related Report
      2004 Annual Research Report
  • [Journal Article] ADP Reduces the Oxygen Binding Affinity of A Sensory Histidine Kinase, FixL : Possibility of the Enhanced Reciprocating Kinase Reaction2004

    • Author(s)
      H.Nakamura, H.Kumita, K.Imai, T.Iizuka, Y.Shiro
    • Journal Title

      Proc.Natl.Acad.Sci.USA 101

      Pages: 2742-2746

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Structural Basis of Human Cytoglobin for Ligand Binding2004

    • Author(s)
      H.Sugimoto, M.Makino, H.Sawai, N.Kawada, K.Yoshizato, Y.Shiro
    • Journal Title

      J.Mol.Biol. 339

      Pages: 873-885

    • Related Report
      2004 Annual Research Report
  • [Journal Article] NO Reduction by Nitric Oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa : Characterization of Reaction Intermediates That Appear in the Single-Turnover Cycle2004

    • Author(s)
      H.Kumita, K.Matsuura, T.Hino, S.Takahashi, H.Hori, Y.Fukumori, I.Morishima, Y.Shiro
    • Journal Title

      J.Biol.Chem. 279

      Pages: 55247-55254

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Design and Synthesis of de Novo Cytochrome c2004

    • Author(s)
      M.Ishida, N.Domae, Y.Shiro, T.Oku, T.Iizuka, Y.Isogai
    • Journal Title

      Biochemistry 43

      Pages: 9823-9833

    • Related Report
      2004 Annual Research Report
  • [Journal Article] D88A Mutant of Cytochrome P450nor Provides Kinetic Evidence for Direct Complex Formation with Electron Donor NADH2004

    • Author(s)
      M.Umemura, F.Su, N.Takaya, Y.Shiro, H.Shoun
    • Journal Title

      Eur.J.Biochem. 271

      Pages: 2887-2894

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Substrate Recognition and Molecular Mechanism of Fatty Acid Hydroxylation by Cytchrome P450 from Bacillus subtilis : Crystallographic, Spectroscopic and Mutational Studies2003

    • Author(s)
      D.-S.Lee, et al.
    • Journal Title

      J.Biol.Chem. 278

      Pages: 9761-9767

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Characterization of Heme Environmental Structure of Cytoglobin, A Fourth Globin in Human2003

    • Author(s)
      H.Sawai, et al.
    • Journal Title

      Biochemistry 42

      Pages: 5133-5142

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] O_2 Specific Regulation of the Ferrous Heme-based Sensor Kinase FixL from Sinorhizobium meliloti and Its Aberrant Inactivation in the Ferric Form2003

    • Author(s)
      S.Akimoto, et al.
    • Journal Title

      Biochem.Biophys.Res.Commun. 304

      Pages: 136-142

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Direct Observation of Photolysis-induced Tertiary Structural Changes in Hemoglobin2003

    • Author(s)
      S.Adachi, et al.
    • Journal Title

      Proc.Natl.Acad.Sci.USA 100

      Pages: 7039-7044

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Synthesis of Biotinylated Heme and Its Application to Panning Heme-binding Proteins2003

    • Author(s)
      M.Ishida et al.
    • Journal Title

      Anal.Biochem. 321

      Pages: 138-141

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Synthesis of Biotinylated Heme and Its Application to Panning Heme-binding Proteins2003

    • Author(s)
      M.Ishida, et al.
    • Journal Title

      Anal.Biochem. 321

      Pages: 138-141

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Enzymatic Reaction of Hydrogen Peroxide-dependent Peroxygenase Cytochrome P450s : Kinetic Deuterium Isotope Effects and Analyses by Resonance Raman Spectroscopy2002

    • Author(s)
      I.Matsunaga, et al.
    • Journal Title

      Biochemistry 41

      Pages: 1886-1892

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Crystallization and Preliminary X-ray Diffraction Analysis of Fatty Acid Hydroxylase Cytochrome P450Bsβ from Bacillus subtilis2002

    • Author(s)
      D.-S.Lee, et al.
    • Journal Title

      Acta Crystal. D58

      Pages: 687-689

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] D.-S.Lee, A.Yamada, H.Sugimoto, I.Matsunaga, H.Ogura, K.Ichihara, S.Adachi, S.-Y.Park, Y.Shiro: "Substrate Recognition and Molecular Mechanism of Fatty Acid Hydroxylation by Cytchrome P450 from Bacillus subtilis : Crystallographic, Spectroscopic and Mutational Studies"J.Biol.Chem.. 278. 9761-9767 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] H.Sawai, N.Kawada, K.Yoshizato, H.Nakajima, S.Aono, Y.Shiro: "Characterization of Heme Environmental Structure of Cytoglobin, A Fourth Globin in Human"Biochemistry. 42. 5133-5142 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] S.Akimoto, A.Tanaka, K.Nakamura, Y.Shiro, H.Nakamura: "O2 Specific Regulation of the Ferrous Heme-based Sensor Kinase FixL from Sinorhizobium meliloti and Its Aberrant Inactivation in the Ferric Form"Biochem. Biophys.Res.Commun.. 304. 136-142 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] S.Adachi, S.-Y.Park, J.R.H.Tame, Y.Shiro, N.Shibayama: "Direct Observation of Photolysis-induced Tertiary Structural Changes in Hemoglobin"Proc.Natl.Acad.Sci.USA. 100. 7039-7044 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] M.Ishida, N.Dohmae, Y.Shiro, Y.Isogai: "Synthesis of Biotinylated Heme and Its Application to Panning Heme-binding Proteins"Anal.Biochem.. 321. 138-141 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] H.Nakamura, H.Kumita, K.Imai, T.Iizuka, Y.Shiro: "ADP Reduces the Oxygen Binding Affinity of A Sensory Histidine Kinase, FixL : Possibility of the Enhanced Reciprocating Kinase Reaction"Proc.Natl.Acad.Sci.USA. 101. 2742-2746 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Matsunaga, I., Sumimoto, T., Ayata, M., Ogura, H.: "Functional modulation of a peroxygenase cytochrome P450 : novel insight into the mechanisms of peroxygenase and peroxidase enzymes"FEBS Letters. 26487. 1-5 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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