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A CO_2 fixation enzme (PEP carboxylase) : Functional analysis by genetic engineering and X-ray chrystallography

Research Project

Project/Area Number 10480157
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

IZUI Katsura  Kyoto University, Graduate School of Biostudies, Professor, その他の独立研究科, 教授 (20025414)

Co-Investigator(Kenkyū-buntansha) KAI Yasushi  Osaka University, Graduate School of Technology, Professor, 工学研究科, 教授 (40029236)
Project Period (FY) 1998 – 2000
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥12,800,000 (Direct Cost: ¥12,800,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥9,700,000 (Direct Cost: ¥9,700,000)
KeywordsCO_2-fixation enzyme / phosphoenolpyruvate carboxylase / X-ray crystallography / protein engineering / site-directed mutagenesis / enzyme reaction mechanism / allosteric regulation mechanism / regulation by phosphorylation / 大腸菌 / トウモロコシ / ホスホエトルピルビン酸カルボキシラーゼ / C4光合成 / 部位特異的変異
Research Abstract

Phosphoenolpyruvate carboxylase (PEPC) is an important CO_2-fixation enzyme. In C4 plants, the enzyme catalyzes the primary CO_2 assimilation reaction for photosynthesis in C4 plants. 1) We elucidated the 3-dimensional structure of PEPC by X-ray crystallography for the first time in the world, and provided a basis for functional analysis by genetic engineering. 2) From the structure of E, coli PEPC liganded with an allosteric inhibitor, aspartate, a unique model for the allosteric inhibition mechanism was proposed. 3) From the structure of E. coli PEPC liganded with PEP analogue, DCDP, the location of catalytic site was unequivocally established. 4) PEPC of Zea mays was crystallized and its 3-dimensional structure was also elucidated. By comparing the structures of PEPCs from E. coli and Zea mays, a plausible model for the reaction mechanism was deduced. 5) Various recombmant maize enzymes having mutations near the regulatory phosphorylation site were prepared and its interaction with a specific protein kinase is under investigation. 6) The site of glucose 6-phosphate, an allosteric activator of maize PEPC, was suggested by crystallographic analysis and the amino acid residues involved were identified by site-directed mutagenesis. 7) A flexible loop bridging over the catalytic site was shown to be indispensable for catalytic avtivity and mainly involved in the binding of another substrate, HCO_3. 8) A cDNA for protein kinase involved in the regulatory phsphorylation of PEPC was cloned from a C4 plant for the first time and characterized.

Report

(4 results)
  • 2001 Final Research Report Summary
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (34 results)

All Other

All Publications (34 results)

  • [Publications] Chen, L.M.: "Molecular characterization of a phosphoenolpyruvate carboxylase from a thermo-philic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties"Plant Cell Physiol.. 43. 159-169 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Saze, H.: "Thioredoxin-mediated reductive activation of a protein kinase for the regulatory phosphorylation of C4-form phosphoenolpyruvate carboxylase from maize"Plant Cell Physiol. (Rapid paper). 42. 1295-1302 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tsuchida, Y.: "Phosphoenolpyruvate carboxylase kinase involved in C4 photosynthesis in Flaveria trinervla : cDNA cloning and characterization"FEBS Lett.. 507. 318-322 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 甲斐 泰: "ホスホエノールピルビン酸カルボキラーゼの3次元構造:そのアロステリック阻害機構"生物物理. 41. 9-14 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Parvathi, K.: "Illumination increases the affinity of phosphoenolpyruvate carboxylase to bicarbonate in leaves of a C4 plant, Amaranthus hypochondriacus"Plant Cell Phsiol.. 41. 95-910 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tanabe, M.: "Production and analysis of transgenic C3-C4 intermediate Moricandia arvensis expressing a maize C4 phosphoenolpyruvate carboxylase gene"Plant Biotechnology. 17. 93-98 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ueno, Y.: "Immunological analysis of the phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised againsy a synthetic phoshorylated peptide"Plant J.. 21. 17-26 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] H.Matsumura, T.: "Crystallization and preliminary X-ray diffraction studies of C4-form phosphoenolpyruvate carboxylase from maize"Acta Crystallogr D Biol Crystallogr.. 55. 1937-1938 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] H.Matsumura, M.: "Plausible phosphoenolpyruvate binding site revealed by 2.6 Astructure of Mn^<2+>-bound phosphoenolpyruvate carboxylase from Escherichia coll"FEBS Lett.. 458. 93-96 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kai, Y.: "Three-dimensional structure of phosphoenolpyruvate carboxylase : A proposed mechanism for allosteric inhibition"Proc. Natl. Acad. Sci. USA. 96. 823-828 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ogawa, N.: "Characterization of a maize Ca^<2+>-dependent protein kinase phosphorylating phosphoenolpyruvate carboxylase"Plant Cell Physiol.. 39. 1010-1019 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Dong, L.-Y.: "Cloning, expression and characterization of a root-form phosphoenolpyruvate carboxylase from Zea mays : Comparison with the C4-form enzyme"Plant Cell Physiol.. 39. 865-873 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Chen L.M.: "Molecular characterization of a phosphoenolpyruvate carboxylase from a thermophilic cyanobacterium, syechococcus vulcanus with unusual allosteric properties"Plant Cell Physiol.. 43. 159-169 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Saze H.: "Thioredoxin-mediated reductive activation of a protein kinase for the regulatory phosphorylation of C4-form phosphoenolpyruvate carboxylase from maize"Plant Cell Physiol. (Rapid paper). 42. 1295-1302 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tsuchida Y.: "Phosphoenolpyruvate carboxylas kinase involved in C4 photosynthesis in Flaveria trinervia : cDNA cloning and characterization"FEBS Lett.. 507. 318-322 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kai Y.: "Three-dimensional structure of phosphonolpyruvate : Mechanism of allosteric inhibition (in Japanese)"Biophysics. 41. 9-14 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Parvathi K.: "Illumination increases the affinity of phosphoenolpyruvate carboxylase to bicarbonate in leaves of a C4 plant, Amaranthus hypochondriacus"Plant Cell Physiol.. 41. 905-910 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tanabe M.: "Production and analysis of transgenic C3-C4 intermediate Moricandia arvensis expressing a maiza C4 phosphoenolpyruvate carboxylase gene"Plant Biotechnology. 17. 905-910 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ueno Y.: "Immunological analysis ofthe phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised againsy a synthetic phosphorylated peptide"Plant J.. 21. 17-26 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Matsumura H.: "Crystallization and preliminary X-ray diffraction studies of C4-form phosphoenolpyruvate carboxylase from maize"Acta Crystallogr D Biol Crystallogr. 55. 1937-1938 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Matsumura H.: "Plausible phosphoenolpyruvate binding site revealed by 2.6 Å structure of Mn^<2+>-bound phosphoenolpyruvate carboxylase from Escherichia coli"FEBS Lett.. 458. 93-96 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kai Y.: "Three-dimensional structure of phosphoenolpyruvate carboxylase : A proposed mechanism for allosteric inhibition"Proc. Natl. Acad. Sci. USA. 96. 823-828 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ogawa N.: "Characterization of a maize Ca^<2+>-dependent protein kinase phosphorylating phosphoenolpyruvate carbixylase"Plant Cell Physiol.. 39. 1010-1019 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Dong L.-Y.: "Cloning, expression and characterization of a root-form phosphoenolpyruvate carboxylase from Zea mays : Comparison with the C4-form enzyme"Plant Cell Physiol.. 39. 365-873 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 甲斐泰: "ホスホエノールピルビン酸カルボキシラーゼの3次元構造:そのアロステリック阻害機構"生物物理. 41・1. 9-14 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] H.Matsumura: "Crystal structure of phosphoenolpyruvate carboxylase : The reaction mechanism and regulation"(発表予定).

    • Related Report
      2000 Annual Research Report
  • [Publications] L.-M.Chen: "Molecular characterization of phosphoenolpyruvate carboxylase from a thermophilic cyano bacterium. Synechococcus vulcanus"(発表予定).

    • Related Report
      2000 Annual Research Report
  • [Publications] M.Kotera: "Phosphoenolpyruvate carboxylase of C_4 photosynthesis : desensitization to glucose b-phosphate by site-directed mutagenesis"(発表予定).

    • Related Report
      2000 Annual Research Report
  • [Publications] A.Terada: "Catalytic role of a flexible role of phosphoenol. pyruvate carboxylase as studied by site-directed mutage nesis"(発表予定).

    • Related Report
      2000 Annual Research Report
  • [Publications] Y.Kai: "Three-dimensional structure of phosphoenolpyruvate carboxylase"Proc. Natl. Acad. Sci. USA. 96・3. 823-828 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] H.Matsumura: "Plausible phosphoenolpyruvate binding site revealed by 2.6 A structure of Mn2+- bound phosphoenolpyruvate carboxylase from Escherichia coli"FEBS Lett.. 458・2. 93-96 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] H.Matsumura: "Crystallization and preliminary X-ray diffraction studies of C4-form phosphoenolpyruvate carboxylase from maize"Acta Cryst.. D55・11. 1937-1938 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K.Tsumura: "Role of flexible loop in catalytic activity of phosphoenolpyruvate carboxylase"発売予定.

    • Related Report
      1999 Annual Research Report
  • [Publications] Kai et al.: "Three dimensional structure of phosphoenolpyruvate carboxy lose:A proposed mechanism for allosteric inhibition" Proc.Natl Acad.Sci.USA. 96・3. 823-828 (1999)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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