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Mechanistic Studies on Precise Substrate Recognition and Product Specificity of 2,3-Oxidosqualene Cyclases

Research Project

Project/Area Number 08458173
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Bioorganic chemistry
Research InstitutionThe University of Tokyo

Principal Investigator

EBIZUKA Yutaka  The University of Tokyo, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学系研究科, 教授 (90107384)

Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥7,300,000 (Direct Cost: ¥7,300,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥4,900,000 (Direct Cost: ¥4,900,000)
Keywords2,3-Oxidosqualene / Cyclase / Lanosterol / Cycloartenol / Triterpene / beta-Amyrin / Lupeol / Chimeric Enzyme / オキシドスクワレン / 大量発現 / cDNA
Research Abstract

The successful cloning of lanosterol synthase cDNA from mammals and cycloartenol synthase cDNA from pea in the previous year led us to try cDNA clonig of triterpene synthases from Panax ginseng hairy roots which accumulate dammarane and oleanane type triterpene saponins in addition to phytosterols. (1) cDNA cloning of oxidosqualene cyclases from Panax ginseng hairy roots. Nested PCRs using two sets of degenerate oligonucleotide primers designed from the conserved sequence found in the known oxidosqualene cyclases amplified fragments of expected size. By utilizing the sequence of these fragments, three independent full length cDNA clones, namely PNX,PNY and PNZ,were isolated by RACE method. Expression of these cDNA in yeast established the translation product of PNX to be cycloartenol synthase and that of PNY to be beta-amyrin synthase while the function of PNZ remained unclear although it is presumed to encode dammarenediol synthase. PNY is the first cDNA ever cloned which encodes triterpene synthase. (2) Construction of chimeric clones of lupeol and beta-amyrin synthases and analysis of their functions. Another triterpene synthase, namely lupeol synthase, cDNA was cloned from Atabidopsis thaliana by PCR using the sequence reported in the data base. These two clones share 70% sequence identity each other indicating the difference of 30% amino acid residue determins the product specificity. In order to identify the region responsible for the product specificity, 16 chimeric clones were constructed and analyzed for their functions to reveal that the region between 260 to 340 amino acid from N-terminus is determing the product specificity.

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Tetsuo, Kushiro.: "In ritro conversion of 2,3-oxidosqnaline into dammarenedid by Panax ginsing microsomes." Biol.Pharm.BUll.20(3). 292-294 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Masayo, Morita: "Molecnlar cloning of pen cDNA encoding cycloartenil synthase and its functional expression in yerst." Biul.Pharm,Bull.20(7). 770-775 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Tetsuo Kushiro, Yoshimi Ohno, Masaaki Shibuya and Yutaka Ebizuka: "In vitro Conversion of 2,3-Oxidosqualene into Dammarenediol by Panax ginseng Microsomes" Biol.Pharm.Bull.20. 292-294 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Masayo Morita, Masaaki Shibuya, Moon-Soon Lee, Ushio Sankawa and Yutaka Ebizuka: "Molecular Cloning of Pea cDNA Encoding Cycloartenol Synthase and Its Functional Expression in Yeast" Biol.Pharm.Bull.20. 770-775 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Tetsuo Kushiro, Masaaki Shibuya and Yutaka Ebizuka: Oxidosqualene cyclases from Panax ginseng hairy roots. (in preparation),

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Tetsuo Kushiro, Masaaki Shibuya and Yutaka Ebizuka: Non-regiospecific deprotonation in lupeol synthase reaction. (in preparation),

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Tetsuo Kushiro, Masaaki Shibuya and Yutaka Ebizuka: Identification of the domain responsible for product specificity in triterpene synthases. (in preparation),

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] S.You, K.Ichinose, K.Inoue, M.Shibuya, X.-S.Yao and Y.Ebizuka: Biosynthetic studies on steroid saponins, in "Towards Natural Medicine Research in the 21st Century", ed. by H.Ageta et. al.Elsevier Science B.V., Amsterdam, (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Tetsuo Kushiro: "In vitro conversion of 2,3-oxidosynclene into dammarenediol by Pannx ginseng microsones" Biol.Pharm.Bull.20(3). 292-294 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Masayo Morita: "Molecular cloning of pea cDNA encoding cycloastenal synthise and its functional expression in yenst" Biol.Pharm.Bull.20(7). 770-775 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Tetsuo KUSHIRO: "In vitro Conversion of 2,3-oxidosqualene into dammarenediol by Panax ginseng microsomes." Biol.Pharm.Bull.20(in press). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Masayo MORITA: "Molecular cloning of pea cDNA encoding cycloartenol synthrse and its functional expression in yeast." Biol.Pharm.Bull.20(in press). (1997)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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