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Designing composite biocatalysts by analysis of the proton-transfer process in pyridoxal enzymes

Research Project

Project/Area Number 13125209
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Science and Engineering
Research InstitutionOsaka Medical College

Principal Investigator

HAYASHI Hideyuki  Osaka Med. Col., Dept. Biochemistry, Associate Professor, 医学部, 助教授 (00183913)

Co-Investigator(Kenkyū-buntansha) YANO Takato  Osaka Med. Col., Dept. Biochemistry, Assistant Professr, 医学部, 講師 (40239827)
KURAMITSU Seiki  Osaka Univ., Graduate School of Science, Professor, 大学院・理学研究科, 教授 (60153368)
KAGAMIYAMA Hiroynki  Osaka Med. Col., Dept. Biochemistry, Professor, 医学部, 教授 (80028555)
MIZUGUCHI Hiroyuki  Osaka Med. Col., Dept. Biochemistry, Research Associate, 医学部, 助手 (40247838)
石井 誠志  大阪医科大学, 医学部, 助手 (10247851)
Project Period (FY) 2001 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥23,600,000 (Direct Cost: ¥23,600,000)
Fiscal Year 2003: ¥8,400,000 (Direct Cost: ¥8,400,000)
Fiscal Year 2002: ¥8,600,000 (Direct Cost: ¥8,600,000)
Fiscal Year 2001: ¥6,600,000 (Direct Cost: ¥6,600,000)
Keywordsproton-transfer / pKa / catalytic-mechanism / free-energy / induced-fit / stereochemistry / transferase / composite-biocatalyst / アミノ基転移酵素 / コンフォメーション / スフィンゴ脂質 / ピリドキサールリン酸 / 歪み
Research Abstract

By analyzing the proton-transfer process of pyridoxal enzymes, we elucidated the refined catalytic reaction mechanisms of them, thereby presenting the molecular understanding of the "induced fit" and "multisubstrate recognition". In aspartate aminotransferase (AAT), the conformational hange to the closed form induces an electrostatic repulsion between the main chain carbonyl group of Gly38 and the Schiff base, thereby raising the ree energy level of the Michaelis complex and increasing the k_<cat> value. Kinetic and structural analysis on the reaction of AAT with C5 substrates in comparison with C4 substrates revealed that in the case of AAT-C5 the Michaelis complex takes the open conformation and the external aldimine complex takes the closed conformation, while in AAT-C4, both complexes take the closed conformation. In the closed conformation, There is a hydrophobic interaction between the residues that is located at the active site entrance and covers the active site after the substrate binding. In the Michaelis complex with C5, the loss of this hydrophobic interaction increases the free energy level of the Michaelis complex, again contributing to the increase in the k_cat Value for C5 substrates. In this way, we revealed the systematic interactions between the parts--catalytic part, recognition part, regulation part, and so on--of composite biocatalysts.

Report

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

    (49 results)

All Other

All Publications (49 results)

  • [Publications] Nakai, Y.: "Yeast Nfs1p is involved in thio-modification of both mitochondrial and cytoplasmic tRNAs."J.Biol.Chem.. 279・13. 12363-12368 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ikushiro, H.: "Reactions of serine palmitoyltransferase with serine and molecular mechanisms of the actions of serine derivatives as inhibitors."Biochemistry. 43・4. 1082-1092 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Matsuda, N.: "Instability of the apo form of aromatic L-amino Acid decarboxylase in vivo and in vitro : implications for the involvement of the flexible loop that covers the active site."J.Biochem.. 135・1. 33-42 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Hosono: "Glutamine : phenylpyruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8."J.Biochem.. 134・6. 843-851 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Islam, M.M.: "Reaction of aspartate aminotransferase with C5-dicarboxylic acids : comparison with the reaction with C4-dicarboxylic acids."J.Biochem.. 134・2. 277-285 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Mizuguchi, H.: "Characterization of histidinol phosphate aminotransferase from Escherichia coli."Biochim.Biophys.Acta. 1647・1-2. 321-324 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ikushiro, H.: "Bacterial serine palmitoyltransferase : a water-soluble homodimeric prototype of the eukaryotic enzyme."Biochim.Biophys.Acta. 1647・1-2. 116-120 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Hayashi, H.: "Strain and catalysis in aspartate aminotransferase."Biochim.Biophys.Acta. 1647・1-2. 103-109 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Goto, M.: "Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate : true reaction intermediate and double substrate recognition of the enzyme."Biochemistry. 42・13. 3725-3733 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Hayashi, H.: "Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis."J.Biol.Chem.. 278・11. 9481-9488 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Olmo, M.T.: "Spectroscopic analysis of recombinant rat histidine decarboxylase."J.Biochem.. 132・3. 433-439 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 林秀行: "酵素反応のエネルギー論"生化学. 74・1. 5-16 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Kagamiyama, H.: "Release of enzyme strain during catalysis reduces the activation energy barrier."Chem.Rec.. 1・5. 385-394 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Nakayama, T.: "Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration."FEBS Lea.. 499・1-2. 107-111 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ikushiro, H.: "A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain, purification, characterization, cloning, and overproduction."J.Biol.Chem.. 276・21. 18249-18256 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Ikushiro, H.Hayashi, H.Kagamiyama: "A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. purification, characterization, cloning, and overproduction."J. Biol. Chem.. 276. 18249-18256 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Nakayama, T.Sato, Y.Fukui, K.Yonekura-Sakakibara, H.Hayashi, Y.Tanaka, T.Kusumi, T.Nishino: "Specificity analysis and mechanism of aurone synthesis catalyzed by aurensidin synthase, a polyphenol oxidase homolog responsible for flower coloration."FEBS Lett.. 499. 107-111 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.kagamiyama, H.Hayashi: "Release of enzyme strain during catalysis reduces the activation energy barrier."Chem. Rec.. 1. 385-394 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] M.T.Olmo, F.Sanchez-Jimenez, M.A.Medina, H.Hayashi: "Spectroscopic analysis recombinant rat histidine decarboxylase."J. Biochem.. 132. 433-439 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Hayashi, H.Mizuguchi, I.Miyahara, Y.Nakajima, K.Hirotsu, H.Kagamiyama: "Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis."J. Biol. Chem.. 278. 9481-9488 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Hayashi, H.Mizuguchi, I.mIyahara, M.M.Islam, H.Ikushiro, Y.Nakajima, K.HIrotsu, H.Kagamiyama: "Strain and catalysis in aspartate aminotransferase."Biochim. Biophys. Acta. 1647. 103-109 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Ikushiro, H.Hayashi, H.Kagamiyama: "Bacterial serine palmitoyltransferase : a water-soluble homodimeric prototype of the eukaryotic enzyme."Biochim. Biophys. Acta. 1647. 116-120 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Mizuguchi, H.Hayashi, I.Miyahara, K.Hirotsu, H.Kagamiyama: "Characterization of histidinol phosphate aminotransferase from Escherichia coli."Biochim. Biophys. Acta. 1647. 321-324 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] M.M.Islam, H.Hayashi, H.Kagamiyama: "Reaction of aspartate aminotransferase with C5-dicarboxylic acids : comparison with the reaction with C4-dicarboxylic acids."J. Biochem.. 134. 277-285 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] A.Hosono, H.Mizuguchi, H.Hayashi, M.Goto, I.Miyahara, K.Hirotsu, H.Kagamiyama: "Glutamine : phenylpyruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8."J. Biochem.. 134. 843-851 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] N.Matsuda, H.Hayashi, S.Miyatake, T.Kuroiwa, H.Kagamiyama: "Instability of the apo form of aromatic L-amino acid decarboxylase in vivo and in vitro : implications for the involvement of the flexible loop that covers the active site."J. Biochem.. 135. 33-42 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Ikushiro, H.Hayashi, H.Kagamiyama: "Reactions of serine palmitoyltransferase with serine and molecular mechanisms of the actions of serine derivatives as inhibitors."Biochemistry. 43. 1082-1092 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Nakai, N.Umeda, T.Suzuki, M.Nakai, H.Hayashi, K.Watanabe, H.Kagamiyama: "Yeast Nfslp is involved in thio-modification of both mitochondrial and cytoplasmic tRNAs."J. Biol. Chem.. 279. 12363-12368 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ikushiro, H.: "Reactions of serine plamitoyltransferase with serine and molecular mechanisms of the actions of serine derivatives as inhibitors."Biochemistry. 43・4. 1082-1092 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Goto, M.: "Crystal structures of glutamine : Phenylpyruvate aminotransferase from Thermus thermophilus HB8: Induced fit and substrate recognition"J.Biol.Chem.(Epub ahead of print : M311575200). 279(in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Nakai, Y.: "Yeast Nfs1p is involved in thio-modification of both mitochondrial and cytoplasmic tRNAs"J.Biol.Chem.(Epub ahead of print : M312448200). 279(in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Matsuda, N.: "Instability of the apo from of aromatic L-amino Acid decarboxylase in vivo and in vitro : implications for the involvement of the flexible loop that covers the active site"J.Biochem.. 135・1. 33-42 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Hosono, A.: "Glutamine : phyenylpuruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8"J.Biochem.. 134・6. 843-851 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Omi, R.: "Crystal structures of threonine synthase from Thermus thermophilus HB8: conformational change, substrate recognition, and mechanism"J.Biol.Chem.. 278・46. 46035-46045 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Islam, M.M.: "Reaction of aspartate aminotransferase with C5-dicarboxylic acids : comparison with the reaction with C4-dicarboxylic acids"J.Biochem.. 134・2. 277-285 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Mizuguchi, H.: "Characterization of histidinol phosphate aminotransferase from Escherichia coli"Biochim.Biophys.Acta. 1647・1-2. 321-324 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Goto, M.: "Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate : true reaction intermediate and double substrate recognition of the enzyme"Biochemistry. 42・13. 3725-3733 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Hayashi, Hideyuki: "Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis"J.Biol.Chem.. 278(11). 9481-9488 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Goto, Masaru: "Crystal structures of branched-chain amino acid aminotransferase in the complex with glutamate and glutarate"Biochemistry. 42(印刷中). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Olmo, Maria.T.: "Spectroscopic analysis of recombinant rat histidine decarboxylase"J.Biochem.. 132(3). 433-439 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Omi, Rie: "Structure of imidazole glycerol phosphate synthase from Thermus thermophilus HB8"J.Biochem.. 132(5). 759-765 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Watanabe, Akira: "Reaction mechanism of alanine racemase from Bacillus stearohermophilus x-ray crystallographic studies of the enzyme bound with N-(5'-phosphopyridoxyl)alanine"J.Biol.Chem.. 277(21). 19166-19172 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Hayashi, Hideyuki: "Strain and catalysis in aspartate aminotransferase"Biochim.Biophys. Acta. (印刷中). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Haruyama, K.: "Structures of Escherichia colt histidinol-phosphate aminotrans ferase and its complexes with histidinolphosphate and N-(5'-phosphopyridoxyl)-L-glutamate : Double substrate recognition of the enzyme"Biochemistry. 40・15. 4663-4644 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Ikushiro, H.: "A water-soluble homodimeric Serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. purification, characterization, cloning, and overproduction"J. Biol. Chem.. 276・21. 18249-18256 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Okada, K.: "Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine : Induced fit and substrate recognition of the enzyme"Biochemistry. 40・25. 7453-7463 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Watanabe, A.: "Reaction mechanism of alanine racemase from Bacillus stearothermophilus : X-ray crystallographic studies of the enzyme bound with N-(5'-phosphopyridoxyl)-alanine"J. Biol. Chem.. 277(in press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Shimotohno A: "Demonstration of the importance and usefulness of manipulating non-active-site residues in protein design"J. Biochem.. 129・6. 943-948 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 林秀行: "酵素反応のエネルギー論"生化学. 74・1. 5-16 (2002)

    • Related Report
      2001 Annual Research Report

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

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