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2000 Fiscal Year Final Research Report Summary

ANALYSIS OF SUBSTRATE RECOGNITION MECHANISM OF ISOPROPYLMALATE DEHYDROGENASE

Research Project

Project/Area Number 10680612
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionOKAYAMA UNIVERSITY

Principal Investigator

TANAKA Hidehiko  FACULTY OF AGRICULTURE, OKAYAMA UNIVERSITY, PROFESSOR, 大学院・自然科学研究科, 教授 (90065912)

Co-Investigator(Kenkyū-buntansha) TAMURA Takashi  FACULTY OF AGRICULUTURE, OKAYAMA UNIVERSITY, ASSOCIATE PROFESSOR, 農学部, 助教授 (40253009)
INAGAKI Kenji  FACULTY OF AGRICULTURE, OKAYAMA UNIVERSITY, PROFESSOR, 農学部, 教授 (80184711)
Project Period (FY) 1998 – 2000
KeywordsISOPROPYLMALATE DEHYDROGENASE / ISOCITRATE DEHYDROGENASE / SUBSTRATE RECOGNITION MECHANISM / COENZYME SPECIFICITY / X-RAY CRYSTALLOGRAPHY
Research Abstract

1) Structure of 3-isopropylmalate dehydrogenase from Thiobacillus ferrooxidans at 2.0 Åresolution.
3-Isopropylmalate dehydrogenase (IPMDH) gene was cloned and sequenced from Thiobacillus ferrooxidans (Tf). We have determined the crystal structures of Tf-IPMDH and the substrate binding mechanism have been solved. IPMDH shows a marked similarity to isocitrate dehydrogenase (ICDH) in its structural framework and catalytic mechanism, and is classified into a unique group of decarboxylating dehydrogenases. The difference between their substrates is the γ moiety attached to 2R-malate.
The structure shows a fully closed conformation and the substrate-binding site is quite similar to that of ICDH except for a region around the γ-isopropyl group. The γgroup is recognized by a unique hydrophobic pocket, which includes Glu88, Leu91 and Leu92. The Cβ and Cγ atoms of Glu88 interact with the γ-isopropyl moiety of 3-isopropylmalate (IPM) and are central to the recognition of substrate.
2) Mutagenesis of … More substrate recognition site of Tf-IPMDH.
We have changed γmoiety recognition site of Tf-IPMDH (Ala72〜Leu92) to that of Ec-ICDH (Pro102〜Val116). We analyzed the substrate specificity of the mutant enzyme. We have been investigated whether the higher-order structure for the mutant enzyme is stabilized because about ten amino acid residues of leuB gene are substituted. In result, The mutant enzyme's loop regions will be distorted slightly, but we expected the structure of the mutant enzyme was stabilized and began on this theme. The mutant enzyme was expressed as soluble protein in E.coli.
3 )Structure of NAD dependent ICDH from Thiobacillus thiooxidans and coenzyme recognition mechanism.
NAD dependent ICDH gene from Thiobacillus thiooxidans (Tt) was cloned and sequenced and the coenzyme recognition mechanism was analyzed. The amino acid sequence had a high degree of similarity to the NADP dependent ICDH and IPMDH.Tt-ICDH is 59.2%, and 22.6% identical to Ec-ICDH and Tf-IPMDH, respectively. It is expected that the tertiary structure of Tt-ICDH is similar to Ec-ICDH and Tf-IPMDH, because the regions composed a deduced secondary structure (α-helix and β-sheet) of Tt-ICDH are much alike.
Tt-ICDH has conserved Asp357 which is significant of the NAD recognition in the coenzyme binding site alignment. On the other hand, Tt-ICDH lacked for a tyrosine residue which was conserved in NADP-ICDH and recognized phosphate of NADP+. Thus, it is revealed that this enzyme has similar coenzyme binding site to that of the IPMDH's. Less

  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Imada,K.,K.Inagaki,H.Matsunami,H.Kawaguchi,H.Tanaka,N.Tanaka,K.Namba: "Structure of 3-Isopropylmalate Dehydrogenase in Complex with 3-Isopropylmalate at 2.0 Å Resolution : the role of Glu88 in the unique substrate-recognition mechanism."Structure. 6. 971-982 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kawaguchi,H.,K.Inagaki,H.Matsunami,Y.Nakayama,T.Tano,H.Tanaka: "Purification and Characterization of 3-Isopuropylmalate Dehydrpgenase from Thiobacillus thiooxidans"J.Biosci.Bioeng.. 90(4). 459-461 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Inoue,H.,K.Inagaki,N.Adachi,T.Tamura,N.Esaki,K.Soda and H.Tanaka: "Role of Tyrosine 114 of L-Methionine γ-Lyase from Pseudomonas putida"Biosci.Biotech.Biochem. 64・11. 2332-2339 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Seow,T.-K.,K.Inagaki,T.Nakamura,R.Maeda,T.Tamura,and H.Tanaka: "Purification and Some Characteristics of a Monomeric Alanine Racemase from an Extreme thermophile, Thermus thermophilus"J.Biosci.Bioeng. 90・3. 344-346 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tamura,T.,Y.Oki,A.Yoshida,T.Kuriyama,H.Kawakami,H.Inoue,K.Inagaki and H.Tanaka: "Noncompetitive, reversible inhibition of aminoacylase-1 by a series of l-α-hydroxyl-and l-α-fluoro-fatty acids; the ligand specificity of Aspergillus oryzae and porcine kidney enzymes"Archives Biochem.Biophys.. 379・2. 261-266 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Lim,L.-C.,T.Tamura,R.Rahim,M.Ali,H.Ghazali,K.Inagaki and H.Tanaka: "A colourimetric screening method for microorganisms that have methionine γ-lyase activity"Asia Pacific J.Mol.Biol.Biotech.. 8・1. 95-97 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Motoshima,M.,K.Inagaki,T.Kumasaka,M.Furuichi,H.Inoue,T.Tamura,N.Esaki,K.Soda,N.Tanaka,M.Yamamoto,and H.Tanaka: "Crystal Structure of the Pyridoxal-5′-phosphate Dependent L-methionine γ-lyase from Pseudomonas putida"J.Biochem.,. 128・3. 349-354 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Imada, K., Kenji Inagaki, Hideyuki Matsunami, Hiroshi Kawaguchi, Hidehiko Tanaka, N.Tanaka, and K.Namba: "Structure of 3-Isopropylmalate Dehydrogenase in Complex with 3-Isopropylmalate at 2.0ÅResolution : the role of Glu88 in the unique substrate-recognition mechanism."Structure. 6. 971-982 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hiroshi Kawaguchi, Kenji Inagaki, Hideyuki Matsunami, Yumi Nakayama, Tatsuo Tano, and Hidehiko Tanaka: "Purification and characterization of 3-isopropylmalate dehydrogenase from Thiobacillus thiooxidans."J.Biosci.Bioeng.. 90. 459-461 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hiroyuki Inoue, Kenji Inagaki, N.Adachi, Takashi Tamura, Nobuyoshi Esaki, kenji Soda and Hidehiko Tanaka: "Role of Tyrosine 114 of L-Methionine γ-Lyase from Pseudomonas putida."Biosci.Biotech.Biochem.. 64. 2332-2339 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Seow Teck Keong, Kenji Inagaki, T.Nakamura, R.Maeda, Takashi Tamura, and Hidehiko Tanaka: "Purification and Some Characteristics of a Monomeric Alanine Racemase from an Extreme thermophile, Thermus thermophilus"J.Biosci.Bioeng.. 90. 344-346 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Takashi Tamura, Yoshiko Oki, A.Yoshida, T.Kuriyama, H.Kawakami, Hiroyuki Inoue, Kenji Inagaki and Hidehiko Tanaka: "Noncompetitive, reversible inhibition of aminoacylase-1 by a series of 1-a-hydroxyl-and 1-a-fluoro-fatty acids ; the ligand specificity of Aspergillus oryzae and porcine kidney enzymes"Archives Biochem. Biophys.. 379. 261-266 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Lim, L.-C., Takashi Tamura, R.Rahim, M.Ali, H.Ghazali, Kenji Inagaki and Hidehiko Tanaka: "A colourimetric screeningmethod for microorganisms that have methionine γ-lyase activity."Asia Pacific J.Mol.Biol.Biotech.. 8. 95-97 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Motoshima, M., Kenji Inagaki, T.Kumasaka, M.Furuichi, Hiroyuki Inoue, Takashi Tamura, Nobuyoshi Esaki, Kenji Soda, N.Tanaka, M.Yamamoto, and Hidehiko Tanaka: "Crystal Structure of the Pyridoxal-5'-phosphate Dependent L-methionine γ-lyase from Pseudomonas putida."J.Biochem.. 128. 349-354 (2000)

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

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Published: 2002-03-26  

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