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極限酵素の環境適応機構の解明

Research Project

Project/Area Number 16041229
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

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

Principal Investigator

金谷 茂則  大阪大学, 大学院・工学研究科, 教授 (30273585)

Co-Investigator(Kenkyū-buntansha) 高野 和文  大阪大学, 大学院・工学研究科, 助教授 (40346185)
Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 2005: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥3,000,000 (Direct Cost: ¥3,000,000)
Keywords超好熱古細菌 / 低温菌 / リボヌクレアーゼH / サチライシン / ペプチジルプロリルシストランスイソメラーゼ / アミロイドペプチド / 結晶構造 / FKBP22 / 酵素 / 蛋白質 / 極限微生物 / 安定性 / 結晶機構
Research Abstract

生体分子の一つである蛋白質のほとんどは水溶液中で働くので、その構造や機能は水との相互作用で決まる。従って、好熱菌や低温菌の酵素がどのような機構で高温環境や低温環境に適応したのかを明らかにすることができれば、水と蛋白質の相互作用に関しても新たな知見の得られることが期待される。本年度は以下の成果を得た。
1.超好熱菌リボヌクレアーゼHIIの安定性が、オスモライト存在下で大きく向上することを見いだした。このことから、超好熱菌の細胞内には様々な安定化因子の存在することが示唆された。また、活性中心変異体の安定性が大きく向上することを明らかにした。このことから、超好熱菌酵素でも活性中心は不安定化されていることが示唆された。さらに、好熱菌リボヌクレアーゼHIIIの結晶構造を決定した。
2.超好熱菌サチライシンは、他の中温菌由来サチライシン同様プロ体からプロペプチドが切断されて活性型の成熟体になる。しかし、これらのサチライシンとは異なり、超好熱菌サチライシンの成熟化にはプロペプチドではなくCa^<2+>が必須であることを明らかにした。また、成熟体の構造がCa^<2+>の結合により大きく変化することを明らかにした。
3.低温菌FKBP22は、ペプチジルプロリルシストランスイソメラーゼ(PPIase)活性部位を含むC末端ドメインと2量体の形成に必要なN末端ドメインから成る。これらのドメインと部分変性した蛋白質の相互作用を解析することにより、N末端ドメインはPPIaseの蛋白質基質との結合に必要であることを明らかにした。
4.アミロイドβペプチド(Aβ_<28-42>)を超好熱菌リボヌクレアーゼHIIのC末端領域に導入した変異体を作製し、その結晶構造を解析した。その結果、野生型酵素ではαヘリックス構造を形成しているC末端領域が、変異体では逆平行βシートに近い構造を形成していることを明らかにした。

Report

(2 results)
  • 2005 Annual Research Report
  • 2004 Annual Research Report
  • Research Products

    (23 results)

All 2006 2005 2004

All Journal Article (23 results)

  • [Journal Article] Structure of amyloid b fragments in aqueous environments.2006

    • Author(s)
      Kazufumi Takano
    • Journal Title

      FEBS J. 273

      Pages: 150-158

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Crystal structure and structure-based mutational analyses of RNase HIII from Bacillus stearothermophilus : a new Type 2 RNase H with TBP-like substrate-binding domain at the N-terminus.2006

    • Author(s)
      Hyongi Chon
    • Journal Title

      J.Mol.Biol. 356

      Pages: 165-178

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Crystal structure of TBP-interacting protein (Tκ-TIP26) and implications for its inhibition mechanism of the interaction between TBP and TATA-DNA.2006

    • Author(s)
      Takahiko Yamamoto
    • Journal Title

      Protein Sci. 15

      Pages: 152-161

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Amyloidogenecity and pitrilysin sensitivity of a lysine-free derivative of amyloid b-peptide cleaved from a recombinant fusion protein.2006

    • Author(s)
      Joel Cornista
    • Journal Title

      J.Biotechnol. (in press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Gene cloning and in vivo characterization of a dibenzothiophene dioxygenase from Xanthobacter polyaromaticivorans.2006

    • Author(s)
      Shin-ichi Hirano
    • Journal Title

      Appl.Microbiol.Biotechnol. (in press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Crystallization and preliminary X-ray diffraction study of thermostable RNase HIII from Bacillus stearothermophilus.2005

    • Author(s)
      Hyongi Chon
    • Journal Title

      Acta Cryst. F61

      Pages: 293-295

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Overproduction and preliminary crystallographic study of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3.2005

    • Author(s)
      Hyongi Chon
    • Journal Title

      Acta Cryst. F61

      Pages: 319-322

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Gene cloning, overproduction, and characterizations of thermolabile alkaline phosphatase from a psychrotrophic bacterium.2005

    • Author(s)
      Yutaka Suzuki
    • Journal Title

      Biosci.Biotechnol.Biochem. 69

      Pages: 364-373

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Protein crystal growth using laser-processed seed crystals.2005

    • Author(s)
      Kengo Takeuchi
    • Journal Title

      Jpn.J.Appl.Phys. 44

      Pages: 3177-3179

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Comparison of dibenzothiophen-degrading bacteria under low oxygen condition.2005

    • Author(s)
      Fumiya Kitauchi
    • Journal Title

      J.Environ.Biotechnol. 4

      Pages: 117-120

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Crystal structure of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3 at 2.20 Å resolution.2005

    • Author(s)
      Hyongi Chon
    • Journal Title

      Proteins 61

      Pages: 685-688

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Importance of a repetitive nine-residue sequence motif for intracellular stability and functional structure of a family I.3 lipase.2005

    • Author(s)
      Clement Angkawidjaja
    • Journal Title

      FEBS Lett. 579

      Pages: 4707-4712

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Binding analysis of a psychrotrophic FKBP22 to a folding intermediate of protein using surface plasmon resonance.2005

    • Author(s)
      Yutaka Suzuki
    • Journal Title

      FEBS Lett. 579

      Pages: 5781-5784

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Phylogenetic analysis of condensation domain in the nonribosomal peptide synthetases.2005

    • Author(s)
      Niran Roongsawang
    • Journal Title

      FEMS Microbiol.Lett. 252

      Pages: 143-151

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Isolation and characterization of Rhodococcus sp. strains TMP2 and T12 that degrade 2,6,10,14-tetramethylpentadecane (pristane) at moderately low temperatures.2005

    • Author(s)
      Kunihiro, N., Haruki, M., Takano, K., Morikawa, M., Kanaya, S.
    • Journal Title

      J.Biotechnol 115

      Pages: 129-136

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Stabilities and activities of the N- and C-domains of FKBP22 from a psychrotrophic bacterium overproduced in E.coli.2005

    • Author(s)
      Suzuki, Y., Takano, K., Kanaya, S.
    • Journal Title

      FEBS Journal 272

      Pages: 632-642

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Identification of single Mn^<2+> binding sites in the mutant proteins of E.coli RNase HI at Glu^<48> and/or Asp^<134> by X-ray crystallography.2005

    • Author(s)
      Tsunaka, Y., Takano, K., Matsumura, H., Yamagata, Y., Kanaya, S.
    • Journal Title

      J.Mol.Biol. 345

      Pages: 1171-1183

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Isolation and characterization of Xanthobacter polyaromaticivorans sp.nov. 127W that degrades polycyclic and heterocyclic aromatic compounds under extremely low oxygen conditions.2004

    • Author(s)
      Hirano, S., Kitauchi, F., Haruki, M., Imanaka, T., Morikawa, M., Kanaya, S.
    • Journal Title

      Biosci.Biotechnol.Biochem. 68

      Pages: 557-564

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Possible involvement of a FKBP family protein from a psychrotrophic bacterium Shewanella sp. SIB1 in cold-adaptation.2004

    • Author(s)
      Suzuki, Y., Haruki, M., Takano, K., Morikawa, M., Kanaya, S.
    • Journal Title

      Eur.J.Biochem. 271

      Pages: 1372-1381

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Mutational and structural-based analyses of osmolyte effect on protein stability.2004

    • Author(s)
      Takano, K., Saito, M., Morikawa, M., Kanaya, S.
    • Journal Title

      J.Biochem.(Tokyo) 135

      Pages: 701-708

    • NAID

      10016200353

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Kinetic robust monomeric protein from a hyperthermophile.2004

    • Author(s)
      Mukaiyama, A., Takano, K., Haruki, M., Morikawa, M., Kanaya, S.
    • Journal Title

      Biochemistry 43

      Pages: 13859-13866

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Cleavage of various peptides with pitrilysin from Escherichia coli: kinetic analyses using β-endorphin and its derivatives.2004

    • Author(s)
      Comista, J., Ikeuchi, S., Haruki, M., Kohara, A., Takano, K., Morikawa, M., Kanaya, S.
    • Journal Title

      Biosci.Biotechnol.Biochem. 68

      Pages: 2128-2137

    • NAID

      130000030954

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Gene cloning and biochemical characterizations of a thermostable ribonuclease HIII from Bacillus stearothermophilus.2004

    • Author(s)
      Chon, H., Nakano, R., Ohtani, N., Haruki, M., Takano, K., Morikawa, M., Kanaya, S.
    • Journal Title

      Biosci.Biotechnol.Biochem. 68

      Pages: 2138-2147

    • NAID

      130000030955

    • Related Report
      2004 Annual Research Report

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Published: 2004-04-01   Modified: 2018-03-28  

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