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2004 Fiscal Year Final Research Report Summary

Structural biology of C4 enzymes related in regulation of phosphoenolpyruvate carboxylase

Research Project

Project/Area Number 15370045
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionOsaka University

Principal Investigator

KAI Yasushi  Osaka University, Graduate School of Engineering, Professor, 大学院・工学研究科, 教授 (40029236)

Co-Investigator(Kenkyū-buntansha) INOUE Tuyoshi  Osaka University, Graduate School of Engineering, Associate Professor, 大学院・工学研究科, 助教授 (20263204)
KANEHISA Nobuko  Osaka University, Graduate School of Engineering, Lecturer, 大学院・工学研究科, 講師 (20177538)
MATSUMURA Hiroyoshi  Osaka University, Graduate School of Engineering, Assistant Professor, 大学院・工学研究科, 助手 (30324809)
Project Period (FY) 2003 – 2004
KeywordsC4 plant / Phosphoenolpyruvate carboxylase / Mechanism of regulation / Structure-function relation / NADP-denendent malic enzyme / PEPC-PK / PEPC mutant K620S
Research Abstract

With respect to CO_2 assimilation, plants are divided into two types called C3 plants and C4 plants. The photorespiration pathway in C3 plants is caused by the oxygenation reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase and the rate of photorespiration in C3 plants is increased under the environment conditions such as water stress and high temperature, which leads to decrease the efficiency of CO_2 assimilation. In contrast, C_4 plants have a unique pathway of atmospheric CO_2 involved in PEPC. PEPC has a high affinity for the relatively inert bicarbonate ion. C_4-form PEPC is abundantly expressed in C_4 plants compared to that in C_3 plants and catalyzes the first committed step for the fixation of atmospheric CO_2 during C_4-photosynthesis. Recently, some trials to introduce C_4 specific genes into C_3 plants have been made to improve the efficiency of CO_2 fixation in C_3 photosynthesis though recombinant DNA techniques. However, it is still difficult to improve the effi … More ciency because the activities of PEPC are regulated by metabolites such as malate and glucose 6-phosphate and covalent modification by reversible phosphorylation. Thus, the molecular mechanism of PEPC and enzymes related in regulation of PEPC are crucial in improvement of the plants.
In this project, the crystallogphic and biochemical studies of PEPC mutant, PEPC specific kinase called PEPC-PK and C4 type NADP dependent malic enzyme(NADP-ME) were performed. First of all, the structure of K620S mutant, which is insensitive to the negative allosteric effector, was solved by X-ray crystallography. The structure revealed the insensitive mechanism of PEPC. Secondary, the glucose 6-phosphate binding site, which is located at the boundary of the dimer interface, has been determined by site-directed mutagenesis using three dimensional structure information (J.Biol.Chem.280,11798-11806(2005).). This study provided both how the positive effector is bound and how PEPC is regulated by the effector. Thirdly, the purification and crystallization of NADP-ME have been done. The C4 specific enzyme produces the malate which is known as the negative allosteric effector of PEPC. Therefore, the molecular mechanism is expected to provide the insight of C4 photosynthesis regulation. The obtained crystal was very thin and small, but it diffracted up to 3 angstrom resolution in SPring-8. The co-crystallization with several kinds of substrate analogue and structural determination are currently in progress. Fourthly the purification and crystallization of PEPC-PK have been performed. The phosphrylatioin of specific site of PEPC by this specific kinase is believed to change the conformation, which convert into enzymatically active PEPC. The highly purified enzyme was crystallized but it is too small for X-ray diffraction measurement. At present the crystallization of PEPC-PK is performed by several kinds of precipitants and salts and so on. At last, the two review papers, which mentioned the molecular mechanisms of regulation and carboxylation of PEPC, are reported (Annu.Rev.Plant Biol.55,69-84(2004)., Arch.Biochem.Biophys., 414,170-179(2003). Less

  • Research Products

    (50 results)

All 2005 2004 2003 Other

All Journal Article (49 results) Book (1 results)

  • [Journal Article] Maize phosphoenolpyruvate carboxylase : Mutations at the putative binding site for glucose 6-phosphate caused desensitization and abolished responsiveness to regulatory phosphorylation2005

    • Author(s)
      Akiko Takahashi-Terada et al.
    • Journal Title

      J.Biol.Chem. (In press)

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Identification of single Mn^<2+> binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography2005

    • Author(s)
      Yasuo Tsunaka et al.
    • Journal Title

      J.Mol.Biol. 345

      Pages: 1171-1183

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Crystallization and preliminary X-ray diffraction study of thermostable RNase HIII from Bacillus2005

    • Author(s)
      Hyongi Chon et al.
    • Journal Title

      Acta Crystallogr F61

      Pages: 293-295

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Overproduction and preliminary crystallographic study of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT32005

    • Author(s)
      Hyongi Chon et al.
    • Journal Title

      Acta Crystallogr F61

      Pages: 319-322

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Structural Evidence of pH-induced Change Potential of Mavicyanin form Cucurbita pepo medullosa (Zucchini)2005

    • Author(s)
      Y.Xie, T.Inoue, et al.
    • Journal Title

      J.Biochem. (In press)

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Maize phosphoenolpyuvate carboxylase : mutations at the putative binding site for glucose 6-phosphate caused desensitization and aboilished responsiveness2005

    • Author(s)
      Akiko Takahashi-Terada, et al.
    • Journal Title

      J.Biol.Chem. (in press)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Identification of single Mn2+ binding sites required for activation of the mutant proteins of E. coli RNase HI at Glu48 and/or Asp134 by X-ray Crystallography2005

    • Author(s)
      Yasuo Tsunaka et al.
    • Journal Title

      J.Mol Biol. 345

      Pages: 1171-1183

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Structual Evidence of pH-induced Change Potential of Mavicyanin form Cucurbita pepo medullosa (Zucchini)2005

    • Author(s)
      Y.Xie, T.Inoue, et al.
    • Journal Title

      J.Biochem. (in press)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Phosphoenolpyruvate carboxylase : A New Era of Structural Biology2004

    • Author(s)
      Katsura Izui, et al.
    • Journal Title

      Annu.Rev.Plant Biol 55

      Pages: 69-84

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Crystallization and preliminary X-ray crystallographic studies of dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151.2004

    • Author(s)
      Y.Xie, T.Inoue, et al.
    • Journal Title

      Acta Cryst. D60

      Pages: 2383-2386

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Two-liquid hanging-drop vapor-diffusion technique of protein crystallization2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L79-L81

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] First Determination of the Inhibitor Complex Structure of Human Hematopoietic Prostaglandin D Synthase2004

    • Author(s)
      T.Inoue, et al.
    • Journal Title

      J.Biochem. 135

      Pages: 279-283

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Protein Crystal Growth with a Two-Liquid System and Stirring Solution2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      J.Synchrotron Rad. 11

      Pages: 121-124

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] A Semiautomatic Protein Crystallization System with Preventing Evaporation of Drops and Surface Sensor of Solution2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L76-L78

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Protein Crystal Processing Using a Deep-UV Laser2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L73-L75

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Pre-Stirring Promotes Nucleation of Protein Crystals2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L243-L246

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] New Approach to Improve X-ray Diffraction Pattern of Protein Crystal Using UV-Laser Ablative Processing2004

    • Author(s)
      H.Kitano, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L297-L299

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] The effects of Solution Stirring on Protein Crystal Growth2004

    • Author(s)
      Mari Yaoi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L686-L688

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Improving the Quality of Protein Crystals Using Stirring Crystallization2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L522-L525

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Generation of protein crystals using a solution-stirring technique.2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L762-L764

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Universal processing technique for protein crystals using a pulsed UV laser.2004

    • Author(s)
      A.Murakami, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L873-L876

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Novel and simple screening methods for protein crystallization by vapor diffusion rate control2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: 6264-6267

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] レーザー照射による蛋白質結晶成長制御2004

    • Author(s)
      安達宏昭, et al.
    • Journal Title

      日本結晶学会誌 46

      Pages: 238-242

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Application of UV-laser Ablation to Detaching Protein Crystal from Growth Vessel2004

    • Author(s)
      H.Kitano, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L1271-L1274

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Effect of Stirring Method on Protein Crystallization2004

    • Author(s)
      M.Yaoi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L1318-L1319

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Membrane Protein Crystallization Using Laser Irradiation2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L1376-L1378

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Control of Protein Crystal Nucleation and Growth Using Stirring Solution2004

    • Author(s)
      A.Niino, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 11

      Pages: L1442-L1444

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] 紫外レーザーを用いた蛋白質結晶の加工2004

    • Author(s)
      北野博史, et al.
    • Journal Title

      日本結晶学会誌 46

      Pages: 292-296

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Phosphoenolpyruvate carboxylase : A New Era of Structural Biology2004

    • Author(s)
      Katsura Izui, et al.
    • Journal Title

      Annu Rev.Plant Biol. 55

      Pages: 68-84

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Crystallization and preliminary X-ray crystallographic studies of dissmilatory nitrite reductase isolated from Hyphomicrobium denitrificans A31512004

    • Author(s)
      Y.Xie, T.Inoue, et al.
    • Journal Title

      Acta Cryst.D 60

      Pages: 2383-2386

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Two-liquid hanging-drop vapor-diffusion technique of Protein crystallization2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L79-l81

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Protein crystal Growth with a Two-Liquid Systems And Stirring Solution2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      J.Synchrotron Rad. 11

      Pages: 121-124

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] A Semiautomatic Protein Crystallization Systems With Preventing Evaporation of Drops and Surfase Sensor of Solution2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L76-L78

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Protein crystal Processing Using a Deep-UV Laser2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L73-L75

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Pre-Stirring Promotes Nucleaction of Protein Crystals2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L243-L246

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] New Approach to Improve X-ray diffraction Pattern Of Protein Crystal Using UV-Laser Ablative Processing2004

    • Author(s)
      H.Kitano, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L297-L299

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] The effects of solution Stirring on Protein Crystal Growth2004

    • Author(s)
      Mari Yaoi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L686-L688

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Improving the Quality of Protein crystals Using Stirring Crystallization2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L522-L525

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Generation of protein crystals using a solution-Stirring technique2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L762-L764

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Universal processing technique for protein crystals Using a pulsed UV laser2004

    • Author(s)
      A.Murakami, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L873-L876

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Novel and simple screening methods for protein Crystallization by vapor diffusion rate control2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: 6264-6267

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Application of UV-Laser Ablation to Detaching Protein Crystal from Growth Vessel2004

    • Author(s)
      H.Kitano, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L1271-L1274

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Effect of stirring Method on Protein Crystallization2004

    • Author(s)
      M.Yaoi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L1318-L1319

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Membrane Protein crystallization Using Laser Irradiation2004

    • Author(s)
      H.Adachi, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 43

      Pages: L1365-L1378

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Control of Protein Crystal nucleation and Growth Using Stirring Solution2004

    • Author(s)
      A.Niino, et al.
    • Journal Title

      Jpn.J.Appl.Phys. 11

      Pages: L1442-L1444

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Phosphoenolpyruvate carboxylase : Three-dimensional structure and molecular mechanisms2003

    • Author(s)
      Yasushi Kai, et al.
    • Journal Title

      Arch.Biochem.Biophys. 414

      Pages: 170-179

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Phosphoenolpyruvate carboxylase : Three-Dimensional structure and molecular mechanisms2003

    • Author(s)
      Yasushi, Kai, et al.
    • Journal Title

      Arch.Biochem.Biophys 414

      Pages: 170-179

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Crystallization and preliminary X-ray diffraction Study of thermostable RNase HIII from Bacilus

    • Author(s)
      Hyongi Chon, et al.
    • Journal Title

      Acta Crystallogr F 61

      Pages: 293-295

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Overproduction and preliminary crystallographic Study of a human kynurenine aminotransferase II Homologue from Pyrococcus horikoshi OT3

    • Author(s)
      Hyongi Chon, et al.
    • Journal Title

      Acta Crystallogr F 61

      Pages: 319-322

    • Description
      「研究成果報告書概要(欧文)」より
  • [Book] 生体高分子化合物の結晶成長(第5版 実験化学講座5、化学実験のための基礎技術)2005

    • Author(s)
      井上豪, 松村浩由, 甲斐泰
    • Total Pages
      278-288
    • Publisher
      丸善(株)
    • Description
      「研究成果報告書概要(和文)」より

URL: 

Published: 2006-07-11  

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