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

Molecular Design for Stabilization of Proteins Based on Knowledge of an Extreme Thermohile

Research Project

Project/Area Number 02403029
Research Category

Grant-in-Aid for General Scientific Research (A)

Allocation TypeSingle-year Grants
Research Field 物質生物化学
Research InstitutionTokyo Institute of Technology

Principal Investigator

OSHIMA Tairo  Tokyo Institute of Technology, Dept.Life Sci.Professor, 生命理工学部, 教授 (60167301)

Co-Investigator(Kenkyū-buntansha) YAMAGISHI Akihiko  Tokyo Institute of Technology, Dept.Life Sci.Assist.Prof., 生命理工学部, 助手 (50158086)
WAKAGI Takayoshi  Tokyo Institute of Technology, Dept.Life Sci.Assist.Prof., 生命理工学部, 助手 (70175058)
Project Period (FY) 1990 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥21,500,000 (Direct Cost: ¥21,500,000)
Fiscal Year 1993: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1992: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1991: ¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1990: ¥7,500,000 (Direct Cost: ¥7,500,000)
KeywordsStabilization of a protein / Evolutionary molecular engineering / Thermus thermophilus / Site-directed mutagenesis / Isopropylmalate dehydrogenase / Chimeric enzyme / Integration host-vector system / Three dimensional structure / タンパク質安定化設計 / 実験室内進化 / 好熱菌 / 耐熱化分子設計 / 耐熱酵素 / 進化分子工学 / 適応の分子機構 / タンパク質の耐熱化 / タンパク質分子設計 / 超好熱菌 / イソプロピルリンゴ酸デヒドロゲナ-ゼ / 好熱性細菌 / 3ーイソプロピルリンゴ酸デヒドロゲナ-ゼ / タンパク質耐熱化設計 / タンパク質熱安定性 / タンパク質工学
Research Abstract

In order to develop molecular design for making proteins heat stable based on konwledge obtained from biochemical and molecular biological studies on thermophiles, stabilization of 3-isopropylmalate dehydrogenase have been attempted. The stabilized mutants were produced by (1) theoretical design, and (2) evolutionary molecular engineering method.
The three dimensional structure of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, was determined at 1.9A resolution. Based on the structure, mutations have been designed to improve the stabilities of mesophile and chimeric isopropylmalate dehydrogenases. For instance, the structure of the thermophile enzyme suggested importance of hydrophobic interactions at the subunit-subunit interface. Residues at the interface of the Escherichia coli enzyme were replaced with more hydrophobic ones, and the mutant showed a higher denaturation temperature than that of the wild type enzyme. The second approach to prepare stabilized proteins is evolutionary molecular engineering. An integration host-vector system was constructed using T.thermophilus as the host. A gene coding a chimeric enzyme was integrated into the chromosome of the host. The transformant thus obtained could not grow at higher temperature than 70゚C in the absence of leucine. However, after a prolonged incubation at 76 or 79゚C,stabilized mutants appeared which produce stabilized isopropylmalate dehydrogenase. The genes for the stabilized enzyme were analyzed. A result from the present study suggested that a protein can be stabilized if an empty gap inside the molecule is filled by substituting with larger amino acid residue (s). Based on this suggestion, the heat stable enzyme of the thermophile was further stabilized by sitedirected mutagenesis.

Report

(5 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • 1991 Annual Research Report
  • 1990 Annual Research Report
  • Research Products

    (63 results)

All Other

All Publications (63 results)

  • [Publications] Tairo OSHIMA: "Stabilization of enzyme proteins : A case study using 3-isopropylmate dehydrogenase" Protein Eng. 6(8). 1003 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Satoshi AKANMA: "Thermostabilization of chimeric 3-isopropylmate dehydrogenase by random mutagenesis" Protein Eng. 6(8). 1011-1012 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kentaro MIYAZAKI: "Tyrosine-139 in Termus thermophilus 3-isopropylmate dehydrogenase in invdved in catalytic function" FEBS Letters. 332. 37-38 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.TAKADA: "Recombination dificient mutants isolated from an extreme thermophile,Thermus thermophilus" Appl.Environ.Microbid. 59. 2737-2739 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kentaro MIYAZAKI: "Kinetic analysis on the Substracte spectficity of 3-isopropylmaiate dehydrogenase." FEBS Letters. 332. 35-36 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] S.KONDO: "A Physical Map of the Sulfuv Dependent Archaebacterium,Sulfolobus acidocaldarius Chramdche" J.Bacteriol. 175. 1532-1536 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Masami ISHIDA and Tairo OSHIMA: "Overexpression of genes of an extreme thermophile,Thermus thermophilus,in Escherichia" J.Bcteriol. 176(9)(in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hiromi KIRINO et al: "Hydrophobic interaction at the subunit inter face contributes to the unusual thermostability of 3-isopropylmate clehydrogenase from an extreme thermophile,Thermus thermophilus." Eur,J.Biochem. (in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kentaro MIYAZAKI et al.: "Chemical modification and sitedirected mutagenesis of Tyr-36 of 3-isopropylmate dehydrogenase from Thermus Thermophilus HB8." Protein Eng.7(1). 99-102 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kentaro MIYAZAKI and Tairo OSHIMA: "Coenzyme specificity of 3-isopropylmate dehydrogenase from Thermus thermophilus HB8" Protein Eng. 7(3). 401-403 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takayoshi WAKAGI,et al.: "An extremely thermostable isoctrate dehydrogenase from sulfolobus acidocaldarius strain 7 : Purification,crystallization,and general properties including coenzyme specificity and the N-terminal amino acid sequence." Biochem 8Life Sci Adv. (12). 57-66 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Toyoki AMANO et al.: "An Ecto Enzyme from Sulfolobus ucidocaldarius strain 7which Catalyzes Hydrolysis of Inoganic Pyrophosphate,ATP.and ADP : Purification and Characterization" J Biochem.144(3). 329-333 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tairo OSHIMA et al.: "Stabilization of enzyme proteins: A case study using 3-isopropylmate,dehydrogenase" Protein Eng. 6(8). 1003 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Masami ISHIDA and Tairo OSHIMA: "Efficient expression of genes from an extreme thermophile Thermus thermophilus,In Escherichia cdi cells" Protein Eng.6(8). 1011 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Yoko HAYASHI et al.: "Analysis of the denaturation process of chimeric 3-isopropylmate dehydrogenese" Protein Eng.6(8). 1012-1013 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kentaro MIYAZAKI and Tairo OSHIMA: "Tyroshine-139 in Thermus thermophilus 3-isopropylmalate dehydrogenase in involved in catalytic function" FEBS Letters. 332(1/2). 37-38 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kentaro MIYAZAKI et al.: "Kinetic analysis on the substrate specificity of 3-isopropylmalate dehydrogenase" FEBS,Letters. 332(1/2). 35-36 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tohru TAKADA,et al.: "Recombination deficient mutants isolated from an extreme thermophile,Thermus thermophilus" Appl.Environ.Microbiol.59(8). 2737-2739 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Katsumi KAKINUMA et al.: "Enantioselective Synthesis of (2R,3S)-3-Alkylmalic Acids,Competent Substrates for 3-Isopropylmalate Dehydrogenase" Biosci.Biotech.Biochem.57(11). 1916-1923 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kentaro MIYAZAKI,et al.: "Molecular Cloning of the Isocitrate Dehydrogenase Gene of an Extreme Thermophile,Thermus thermophilus HB8" Appl,Environ.Microbiol.58(1). 93-98 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Masahiro SAKURAI,et al.: "Crystallization and preliminary X-ray studies of a Bacillus subtilis and Thermus thermophilus HB8 chimeric 3-isopropylmalate dehydrogenase and thermostable mutants of it" J.Biochem.112(2). 173-174 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takayoshi WAKAGI,et al.: "An extremely stable inorganic pyrophosphatase purified from the cytosol of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius strain 7" Biochim.Biophys.Acta. 1120(2). 289-296 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Katsumi IMADA,et al.: "Three-dimensional Structure of a llighly Thermostable Enzyme. 3-isopropylmalate Dehydrogenase of Thermus thermophilus at 2.2A Resolution" J.Mol.Biol. 222(22). 725-738 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Satoru KANDO Akihiko YAMAGISHI Tairo OSHIMA: "Positive Selection for Uracil Auxotrophs of the Sulfur-Dependent Thermophilic Archaebacterium,Sulfolobus acidocaldarius by Use of 5-Fluoroorotic Acid" J.Bacteriol.173(23). 7698-7700 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tomomi,FUJII et al.: "Crystallization and Preliminary X-Ray Studies on Sulfolobus acidocaldarins Fercedoxin" J.Biochem.110(4). 472-473 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hiromi KIRINO and Tairo OSHIMA: "Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase gene (leuB) from an extreme thermophile,Thermus aguaticus YT-1" J.Biochem.109(6). 852-857 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Ko ONODERA,et al.: "Crystallization and Preliminary X-ray Studies of Chimeric 3-Isopropylmalate Dehydrogenase between Bacillus subtilis and Thermus thermophilus HB8" J.Biochem.109(1). 1-2 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Jing XU,et al.: "Phosphoenolpyruvate-Insensitive Phosphofructokinase Isozyme from Thermus thermophilus HB8" J.Biochem.109(2). 199-203 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kentaro MIYAZAKI,et al.: "Kinetic analysis on the substrate specificity of 3-isopropylmalate dehydrogenase." FEBS Letters. 332 (1/2). 35-36 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tohru TAKADA,et al.: "Recombination deficient mutants isolated from an extreme thermophile, Thermus thermophilus." Appl.Environ.Microbiol.59 (8). 2737-2739 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Katsumi KAKINUMA,et al.: "Enantioselective Synthesis of (2R,3S)-3-Alkylmalic Acids, Competent Substrates for 3-Isopropylmalate Dehydrogenase." Biosci.Biotech.Biochem.57 (11). 1916-1923 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kentaro MIYAZAKI,et al.: "Molecular Cloning of the Isocitrate Dehydrogenase Gene of an Extreme Thermophile, Thermus thermophilus HB8." Appl.Environ.Microbiol.58 (1). 93-98 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Masahiro SAKURAI,et al.: "Crystallization and preliminary X-ray studies of a Bacillus subtilis and Thermus thermophilus HB8 chimeric 3-isopropylmalate dehydrogenase and thermostable mutants of it." J.Biochem.112 (2). 173-174 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takayoshi WAKAGI,et al.: "An extremely stable inorganic pyrophosphatase purified from the cytosol of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius strain 7." Biochim.Biophys.Acta.1120 (2). 289-296 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Katsumi IMADA,et al.: "Three-dimensional Structure of a Highly Thermostable Enzyme, 3-Isopropylmalate Dehydrogenase of Thermus thermophilus at 2.2A Resolution." J.Mol.Biol.222 (22). 725-738 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Satoru KONDO,et al.: "Positive Selection for Uracil Auxotrophs of the Sulfur-Dependent Thermophilic Archaebacterium, Sulfolobus acidocaldarius by Use of 5-Fluoroorotic Acid." J.Bacteriol.173 (23). 7698-7700 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tomomi FUJII,et al.: "Crystallization and Preliminary X-Ray Studies on Sulfolobus acidocaldarius Ferredoxin." J.Biochem.110 (4). 472-473 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hiromi KIRINO,and Tairo OSHIMA: "Molecular cloning and nucleotide sequence of 3-isopropylamalate dehydrogenase gene (1euB) from an extreme thermophile, Thermus aquaticus YT-1." J.Biochem.109 (6). 852-857 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Ko ONODERA,et al.: "Crystallization and Preliminary X-ray Studies of Chimeric 3-Isopropylmalate Dehydrogenase between Bacillus subtilis and Thermus thermophilus HB8." J.Biochem.109 (1). 1-2 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.OSHIMA,K.NUMATA,Y.HAYSHI,H.KIRINO 他: "Stabilization of enzyme proteins:A case study using 3-isopropylmalate dehydrogenase" Protein Eng.6(8). 1003 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] S.AKANUMA,A.YAMAGISHI,T.OSHIMA: "Thermostabilization of chimeric 3-isopropylmalate dehydrogenase by random mutagenesis" Protein Eng.6(8). 1011-1012 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.MIYAZAKI,T.OSHIMA: "Tyrosine-139 in Thermus thermophilus 3-isopropylmalate dehydrogenase is involved in catalytic function" FEBS Letters.332. 37-38 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] T.TAKADA,S.AKANUMA,T.KOTSUKA,M.TAMAKOSHI 他: "Recombination dficient mutants isolated from an extreme thermophile,Thermus thermophilus" Appl.Environ.Microbiol. 59. 2737-2739 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.MIYAZAKI,K.KAKINUMA,H.TERASAWA,T.OSHIMA: "Kinetic analysis on the substracte specificity of 3-isopropylmalate dehydrogenase" FEBS Letters.332. 35-36 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] S.KOND,A.YAMAGISHI,T.OSHIMA: "A Physical Map of the sulfur-Dependent Archaebacterium,Sulfolobus acidocaldarius 7 Chromosome" J.Bacteriol. 175. 1532-1536 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 桐野 裕美,山峯 明彦: "酵素の耐熱化設計" 蛋白質・核酸・酵素. 37. 335-345 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Katsumi IMADA: "Thtee-dimensional structure of a Highly Thermostable Enzyne,3-Isopropylmate Dehydrogenase of Therms thermophilus at 2.2A Resolution" J.Mol.Biol.222. 725-738 (1991)

    • Related Report
      1992 Annual Research Report
  • [Publications] Satoru KONSO: "Positive Selection for Uracil Auxotrophs of the Sulfur-Depensent Thermphilic Archaebacterium,Sulfolobus acidocarius by Use of 5-Fluoroorotic Acid" J.Bacteriol.173. 7698-7700 (1991)

    • Related Report
      1992 Annual Research Report
  • [Publications] Kentaro MIYAZAKI: "Molecular cloning of the isocitrate dehydrogenase gene of an extreme theomophile,Thermus thermophilus HBB" Appl.Environ.Mocrobiol.58. 93-98 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Masahiro SAKURAI: "Crytallization and preliminary X-ray studies of a Bacillus subtilis and Thermus thermophilus HBB chimeric 3-isopropylmalate dehydrogenase and thermostable mutants of it" J.Biochem. 112. 173-174 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Imada,K.: "Threeーdimensional structure of a highly thermostable enzyme,3ーisopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution." J.Mol.Biol.222. 725-738 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Miyazaki,K.: "Molecular Cloning of the Isocitrate Dehydrogenase Gene of an Extreme Thermophile,Thermus thermophilus HB8." Appl.Environ.Microbiol.58. 93-98 (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] Kondo,S.: "Positive selection for uracil auxotrophs in the sulfur dependent thermophilic archaebacterium Sulfolobus acidocaldarius using 5ーfluoroorotic acid." J.Bacteriol.173. 7698-7700 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Onodera,K.: "Crystallization and Preliminary Xーray Studies of Chimeric 3ーIsopropylmalate Dehydrogenase between Bacillus subtilis and Thermus thermophilus HB8." J.Biochem.109. 1-2 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Kirino,H.: "Molecular cloning and nucleotide sequence of 3ーisopropylmalate dehydrogenase gene(leuB)from an extreme thermophile,Thermus aquaticus YTー1." J.Biochem.109. 852-857 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] 桐野 裕美: "酵素の耐熱化設計" 蛋白質核酸酵素. 37. 335-345 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Oshima,T. (S.Osawa and T.Honjo,eds.): "Early Biochemical Evolution:Speculations on the Biochemistry of Primitive Life;in Evolution of Life:Fossils,Molecles,and Culture" Springer Verlag,(Tokyo,Berlin,Heiderberg,New York,London,Paris,HongーKong,Barcelna), 460 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Yamada,T.: "Purification,catalytic properties and thermal stability of threoー3ーisopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile,Thermus thermophilus strain HB8." J.Biochem.108. 449-456 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Kirino,H.: "The DNA Sequence of the 3ーIsopropylmalate Dehydrogenase Gene(leuB)from an Extreme Thermophile,Thermus thermophilus HB8;A Correction."

    • Related Report
      1990 Annual Research Report
  • [Publications] Onodera,K.: "Crystallization and Preliminary Xーray Studies of Chimeric 3ーIsopropylmalate Dehydrogenase between Bacillus subtilis and Thermus thermophilus HB8." J.Biochem.109. 1-2 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Kirino,H.: "Molecular Cloning and Nucleotide Sequence of Isopropylmalate Dehydrogenase Gene(leuB)from an Extreme Thermophile,Thermus aquaticus YTー1."

    • Related Report
      1990 Annual Research Report
  • [Publications] Miyazaki,K.: "Structure of Isocitrate Dehydrogenase Gene of an Extreme Thermophile,Thermus thermophilus HB8."

    • Related Report
      1990 Annual Research Report
  • [Publications] Ikehara,M.(M.Ikehara,K.Titani and T.Oshima,eds.): "Protein Engineering:Protein Design in Basic Research,Medicine,and Industry" Center for Academic Societies Press/Springer Verlag,Tokyo, 355 (1990)

    • Related Report
      1990 Annual Research Report

URL: 

Published: 1990-04-01   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi