• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Reactivity Control Mechanism and Engineering Novel Reactivity in Flavoenzymes

Research Project

Project/Area Number 13125206
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Science and Engineering
Research InstitutionKUMAMOTO UNIVERSITY

Principal Investigator

MIURA Retsu  Kumamoto Univ., Graduate School of Medical Sciences, Professor, 大学院・医学薬学研究部, 教授 (70093466)

Co-Investigator(Kenkyū-buntansha) TAMAOKI Haruhiko  Kumamoto Univ., Graduate School of Medical Sciences, Instructor, 大学院・医学薬学研究部, 助手 (80264290)
SETOYAMA Chiaki  Kumamoto Univ., Graduate School of Medical Sciences, Assoc. Prof., 大学院・医学薬学研究部, 助手 (60040250)
NISHINA Yasuzo  Kumamoto Univ., Graduate School of Medical Sciences, Assoc. Prof., 大学院・医学薬学研究部, 助教授 (50112553)
Project Period (FY) 2001 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥30,000,000 (Direct Cost: ¥30,000,000)
Fiscal Year 2003: ¥9,400,000 (Direct Cost: ¥9,400,000)
Fiscal Year 2002: ¥9,400,000 (Direct Cost: ¥9,400,000)
Fiscal Year 2001: ¥11,200,000 (Direct Cost: ¥11,200,000)
Keywordsflavoenzymes / D-amino acid oxidase / acyl-CoA oxidase / acyl-CoA dehydrogenase / artificial flavins / X-ray crystallography / density functional theory method (8) / 中鎖アシルCoAデヒドロゲナーゼ / 酵素-基質複合体 / 分子軌道計算 / 電荷移動相互作用 / 遷移状態アナログ / 3-チアオクタノイルCoA / 酸素との反応 / 水素結合 / 部位特異的変異
Research Abstract

The three-dimensional structure of acyl-CoA oxidase was solved by X-ray crystallography for the first time. This structure has been long-sought with the expectation of clarifying the mechanism of flavin-oxygen reaction. The detailed structure of the active site provided structural basis of control mechanism of its reaction with molecular oxygen.
On the basis of the three-dimensional structure of D-amino acid oxidase (DAO), the substrate recognition site was mutated by genetic engineering and novel enzymes with capability of oxidizing acidic D-amino acids, which the wild type DAO cannot oxidize, were obtained.
The three-dimensional structure of 3-thiaoctanoyl-CoA-acyl-CoA dehydrogenase (MCAD) complex was solved by X-ray crystallography. This complex has been assigned as a transition-state analog by means of various experimental methods. The crystal structure provided unique information on the interaction between 3-thiaoctanoyl-CoA and the flavin ring. This interaction was verified theoretically by the density functional theory calculations. The results from the calculation was extended to the normal reaction between substrate and enzyme and revealed the important role of the substrate-flavin interaction in enhancing the catalytic process of MCAD.
Artificial flavins with different redox potential were incorporated to FAD bound to MCAD and its reactivity was changed. When 8-cyano-flavin was used as an artificial flavin, MCAD so obtained gained novel reactivity to dehydrogenate propionyl-CoA and 3-ketooctanoyl-CoA, both of which native MCAD is incapable of dehydrogenating. This novel reactivity was obtained by the high redox potential of 8-cyano-flavin compared to native flavin.

Report

(4 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • 2001 Annual Research Report
  • Research Products

    (35 results)

All Other

All Publications (35 results)

  • [Publications] R.Miura, C.Setoyama, Y.Nishina, K.Shiga, I.Miyahara, H.Mizutani, K.Hirotsu: "Porcine kidney D-amino acid oxidase : the three-dimensional structure and its catalytic mechanism based on the enzyme-substrate complex model."J.Mol.Catalysis B : Enzymatic. 12. 43-52 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] R.Miura: "Versatility and specificity in flavoenzymes : Control mechanisms of flavin reactivity."The Chemical Record. 1. 183-194 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nakajima, I.Miyahara, K.Hirotsu, Y.Nishina, K.Shiga, C.Setoyama, H.Tamaoki, R.Miura: "X-Ray crystallographic study of rat acyl-CoA oxidase II."Protein Sci.. 10. 85-86 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nakajima, I.Miyahara, K.Hirotsu, Y.Nishina, K.Shiga, C.Setoyama, H.Tamaoki, R.Miura: "Crystallization and preliminary X-ray characterization of rat liver acyl-CoA oxidase"Acta Crystallogr.. D57. 1680-1681 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] C.Setoyama, Y.Nishina, H.Tamaoki, H.Mizutani, I.Miyahara, K.Hirotsu, K.Shiga, R.Miura: "Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme D-amino acid oxidase. The catalytic and structural roles of Gly313 and Thr317."J.Biochem.. 131. 59-69 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nakajima, I.Miyahara, K.Hirotsu, Y.Nishina, K.Shiga, C.Setoyama, H.Tamaoki, R.Miura: "Three-dimensional structure of the flavoenzyme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase."J.Biochem.. 131. 365-374 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] A.Satoh, Y.Nakajima, I.Miyahara, K.Hirotsu, T.Tanaka, Y.Nishina, K.Shiga, H.Tamaoki, C.Setoyama, R.Miura: "Structure of the transition state analog of medium-chain acyl-COA dehydrogenase. Crystallographic and molecular orbital studies on the charge-transfer complex of medium-chain acyl-CoA dehydrogease with 3-thiaoctanoyl-CoA."J.Biochem.. 134. 297-304 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nishina, K.Sato, H.Tamaoki, H., Tanaka, T., Setoyama, C., R.Miura, K.Shiva: "Molecular mechanism of the drop in the pKa of a substrate analog bound to medium-chain acyl-CoA dehydrogenase : Implications for substrate activation."J.Biochem.. 134. 835-842 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] K.Sato, Y.Nishina, K.Shiga: "Purification of electron-transferring flavoprotein from Megasphaera elsdenii and binding of additional FAD with an unusual absorption spectrum."J.Biochem.. 134. 719-729 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] K.Sato, Y.Nishina, K.Shiga, F.Tanaka: "Hydrogen-bonding dynamics of free flavins in benzene and FAD in electron-transferring flavoprotein upon excitation."J.Photochem.Photobiol.B. 70. 67-73 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 三浦洌: "フラビン酵素における反応制御の構造的基盤"ビタミン. 77. 313-320 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] R.Miura, C.Setoyama, Y.Nishina, K.Shiga, I.Miyahara, H.Mizutani, K.Hirotsu: "Porcine kidney D-amino acid oxidase : the three-dimensional structure and its catalytic mechanism based on the enzyme-substrate complex model"J. MOl. Catalysis B : Enzymatic. 12. 43-52 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] R.Miura: "Versatility and specificity in flavoenzymes : Control mechanisms of flavin reactivity."The Chemical Record. 1. 183-194 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nakajima, I.Miyahara, K.Hirotsu, Y.Nishina, K.Shiga, C.Setoyama, H.Tamaoki, R.Miura: "X-Ray crystallographic study of rat acyl-CoA oxidase II."Protein Sci.. 10, Suppl. 2. 85-86 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nakajima, I.Miyahara, K.Hirotsu, Y.Nishina, K.Shiga, C.Setoyama, H.Tamaoki, R.Miura: "Crystallization and preliminary X-ray characterization of rat liver acyl-CoA oxidase."Acta Crystallogr.. D57. 1680-1681 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nishina, K.Sato, R.Shi, C.Setoyama, R.Miura, K.Shiga: "On the ligands in charge-transfer complexes of porcine kidney flavoenzyme D-amino acid oxidase in three redox states : A resonance Raman study."J. Biochem.. 130. 637-647 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] C.Setoyama, Y.Nishina, H.Tamaoki, H.Mizutani, I.Miyahara, K.Hirotsu, K.Shiga, R.Miura: "Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme D-amino acid oxidase. The catalytic and structural roles of Gly313 and Thr317."J. Biochem.. 131. 59-69 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nakajima, I.Miyahara, K.Hirotsu, Y.Nishina, K.Shiga, C.Setoyama, H.Tamaoki, R.Miura: "Three-dimensional structure of the flavoenzyme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase."J. Biochem.. 131. 365-374 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] A.Satoh, Y.Nakajima, I.Miyahara, K.Hirotsu, T.Tanaka, Y.Nishina, K.Shiga, H.Tamaoki, C.Setoyama, R.Miura: "Structure of the transition state analog of medium-chain acyl-CoA dehydrogenase. Crystallographic and molecular orbital studies on the charge-transfer complex of medium-chin acyl-CoA dehydrogease with 3-thiaoctanooyl-CoA."J. Biochem.. 134. 297-304 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nishina, K.Sato, H.Tamaoki, H., Tanaka, T., Setoyama, C., R.Miura, K.Shiga: "Molecular mechanism of the drop in the pKa of a substrate analog bound to medium-chain acyl-CoA dehydrogenase : Implications for substrate activation."J. Biochem.. 134. 835-842 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yasuzo Nishina: "Molecular mechanism of the drop in the pKa of a substrate analog bound to medium-chain acyl-CoA dehydrogenase : Implications for substrate activation."Journal of Biochemistry. 134. 835-842 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Atsuko Satoh: "Structure of the transition state analog of medium-chain acyl-CoA dehydrogenase.Crystal structure and molecular orbital studies on the charge-transfer complex of medium-chain acyl-CoA dehydrogenase"Journal of Biochemistry. 134. 297-304 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 三浦洌: "フラビン酵素における反応制御の構造的基盤"ビタミン. 77. 313-320 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 三浦洌: "フラビン酵素と酸素の接点.オキシダーゼとデヒドロゲナーゼの選別"化学と生物. 42. 120-126 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Jung-Ja p.Kim: "Acyl-CoA dehydrogenases and acyl-CoA oxidases.Structural basis for mechanistic similarities and differences"European Journal of Biochemistry. 271. 483-493 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Chiaki Setoyama: "Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme D-amino acid oxidase"Journal of Biochemistry. 131. 59-69 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Yoshitaka Nakajima: "Three-dimensional structure of the flavoenzyme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase"Journal of Biochemistry. 131. 365-374 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 鏡山博行: "補酵素作用のダイナミズム-補酵素の機能を支えるアポタンパク質の働き-"ビタミン. 76・5-6. (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Yoshitaka Nakajima: "Structural comparison of acyl-CoA oxidase-II from rat liver with medium-chain acyl-CoA dehydrogenase from pig liver"Flavins and Flavoproteins 2002 (S.Chapman, R.Perham, N.Scrutton, eds.) Rudolf Weber Agency for Scientific Publications, Berlin. 57-62 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Retsu Miura: "Porcine kidney D-amino acid oxidase : the three-dimensional structure and its catalytic mechanism based on the enzyme-substrate complex"Journal of Molecular Catalysis B : Enzymatic. 12. 43-52 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Retsu Miura: "Versatility and specificity of flavoenzymes : Control mechanism of flavin reactivity"The Chemical Record. 1. 183-194 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Yoshitaka Nakajima: "X-Ray crystallographic study of rat acyl-CoA oxidase II"Protein Science. 10 Suppl.2. 85-86 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Yoshitaka Nakajima: "Crystallization and preliminary X-ray characterization of rat liver acyl-CoA oxidase"Acta Crystallographica. D57. 1680-1681 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Yasuzo Nishina: "On the ligands in charge-transfer complexes of porcine kidney flavoenzyme D-amino acid oxidase in three redox states"Journal of Biochemistry. 130. 637-647 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Chiaki Setoyama: "Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme D-amino acid oxidase"Journal of Biochemistry. 131. 59-69 (2002)

    • Related Report
      2001 Annual Research Report

URL: 

Published: 2001-04-01   Modified: 2018-03-28  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi