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Enzyme mechanism analysis of UDP-glucose pyrophosphorylase by X-ray crystal analysis

Research Project

Project/Area Number 09680591
Research Category

Grant-in-Aid for Scientific Research (C)

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

Principal Investigator

KUSUNOKI Masami  Institute for Protein Research, Osaka University, Associate Professor, 蛋白質研究所, 助教授 (90135749)

Co-Investigator(Kenkyū-buntansha) TANIZAWA Katsuyuki  Institute of Scientific and Industrial Research Osaka University, Professor, 産業科学研究所, 教授 (20133134)
Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
KeywordsEnzyme / X-ray Analysis / UDP / Crystal / 反応機構
Research Abstract

UDP-glucose pyrophosphorylase catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose-1-phosphate and MgUTP.We isolated and purified cDNA encoding UDP-glucose pyrophosphorylase from potato tuber.It has 477 amino acid residues.The enzyme was crystallized by the hanging-drop vapor diffusion method with a precipitant of ammonium sulfate.The space group is P212121 with cell dimensions a=108.2, b=124.7, c=87.1 and VM=2.8 /dalton.The crystal structure was solved by multiple-isomorphous replacement with four heavy atom derivatives, K2Pt(CN)4, Hg(CH3COO)2, UO2(NO3)2 and Sm2(SO4)3.The intensity data were collected by an imaging plate diffractometer, RIGALU RAXIS IIc.
The average figure of merit at 3.0 resolution is 0.43, using "mlphare" in CCP4 program package.The phase improvement was carried out by "dm" in CCP4, resulting in a 2.2 resolution density map.The density modification includes histogram-mapping, constraint of Sayer formula, non-crystallographic averaging and solvent flattening, with assumption of 41% solvent content.The final free-R-value was 0.27 at 2.2 resolution.
Two identical molecules are in the crystallographic asymmetric unit.The two molecules are related by 143 degree rotation around an axis almost parallel to the crystallographic z axis with some translation.Each molecule consists of two domains.The N-terminal domain has five helices and seven b-strands (a/b structure) with two additional long helices in N-terminus.The C-terminal domain is mainly composed of left-handed b-helix similar to the structure of UDP-N-acetylglucosamine acyltransferase.The protein structure has been refined by the program X-PLOR.

Report

(3 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report
  • Research Products

    (6 results)

All Other

All Publications (6 results)

  • [Publications] M.Kusunoki,Y.Kitagawa et al: "Left-Handed B Helix Protein,UDP-Glucose Pyrophos phoiylase" Acta Cryst A. S52. C110-C110 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] 楠木正巳,北川康行 他: "UDPグルコースピロホスホリラーゼの結晶構造と活性部位" 日本生化学誌. 68. 647-647 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Kusunoki M.Kitagawa H.Naitou Y.Katsube Y.Sakamoto K.Tanizawa T.Fukui: "LEFT-HANDED b-HELIX PROTEIN UDP-GLUCOSE PYROPHOSPHORYLASE" Acta Cryst A. S52. C110 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Kusunoki M.Kitagawa H.Naitou Y.Katsube Y.Sakamoto K.Tanizawa T.Fukui: "X-ray structure and the active site of UDP-glucose pyrophosphorylase" Seikagaku. 68. 647 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Kusumoto,Y.Kitagawa,H.Katsube,K.Tanigawa et al.: "Left-Handed B-ltelix Protein UDP-Glucose Pyraphosphory lase" Acta Cryst A. S52. C110-C110 (1996)

    • Related Report
      1998 Annual Research Report
  • [Publications] 楠本正巳,北川康行,谷沢克行 他: "UDPグルコースピロホスホリラーゼの結晶構造C活性部位" 日本生化学会誌. 68. 647-647 (1996)

    • Related Report
      1998 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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