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Construction of plant oxygenase libraries and their functional characterization

Research Project

Project/Area Number 18580091
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied biochemistry
Research InstitutionKyoto University

Principal Investigator

MIZUTANI Masaharu  Kyoto University, Institute for Chemical Research, Assistant Professor (60303898)

Project Period (FY) 2006 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
KeywordsCytochrome P450 / Flavin monooxygenase / 2-oxoglutarate dependent dioxygenase / Oxygenase / Arabidopsis / ジオキシゲナーゼ / オキシゲナーゼ
Research Abstract

The aim of this study was to establish the protein libraries of plant oxygenases consisiting of cytochrome P450monooxygenases,2-oxoglutarate dependent dioxygenases, and flavin monooxygenases and to elucidate their biochemical functions.
(1) Plant Monooxygenase Database: The gene sequences as well as the deduced amino acid sequences of three monooxygenases (P450/FMO/2OGD) were extracted from the genome database of Arabidopsis. The phylogenetic analyses of the protein sequences were performed. In addition, the data of the gene expression in various treatments were extracted from MPSS and TAIR database.
(2) Isolation of full ORFs and expression of the recombinant proteins: The full-length cDNAs of P450/FMO/2OGD have been collected from RIKEN-BRC, and the full-ORFs of the other clones were amplified and isolated by RT-PCR approach. After all, about 70% of total oxygenase ORFs have been isolated. The ORFs of FMOs and2OGDs were introduced into the expression vector pETs, and the recombinant pr … More oteins with N-terminal His-tag were expressed in E. coli and were purified with affinity chromatography.
(3) Construction of the co-expression system of plant P450s and P450-reductase; P450s require the electron-transfer from P450-reductase (CPR) for the P450 activities, but E. coli does not have the reductase. The co-expression system of P450s and CPR was constructed with the co-transformation of the two plasmids (pCWori-P450 and pACYC-CPR) into the bacterial cells. The co-expression system enabled us to detect P450 activities in in vitro and in vivo assays in E. coli.
(4) Analysis of At3g13610: We previously found that Arabidopsis roots accumulate large amounts of scopolin, a β-glucoside of coumarin derivtive scopoletin. At3g13610, which is one of the 2OGD genes and exclusively expressed in roots, was found to catalyze ortho-hydroxylation of feruloyl-CoA thioester to form scopoletin. Thus, At3g13610 is Feruloyl-CoA 6'-hydroxylase in Arabidopsis, and this is an ortho-hydrozylase, which is a key enzyme in coumarins biosynthesis in hiaher plants. Less

Report

(3 results)
  • 2007 Annual Research Report   Final Research Report Summary
  • 2006 Annual Research Report
  • Research Products

    (21 results)

All 2008 2007 2006

All Journal Article (19 results) (of which Peer Reviewed: 6 results) Presentation (2 results)

  • [Journal Article] Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl-CoA by an 2-oxoglutarate dependent dioxygenase in Arabidopsis thaliana.2008

    • Author(s)
      Kai K, Mizutani M, Kawamura N, Yamamoto R, Tamai M, Yamaguohi H, Sakata K, Shimizu B
    • Journal Title

      The Plant Journal 55

      Pages: 989-999

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl-CoA by an 2-oxoglutarate dependent dioxygenase in Arabidopsis thaliana2008

    • Author(s)
      Kai, K., Mizutani, M., Kawamura, N., Yamamoto, R., Tamai, M., Yamaguchi, H., Sakata, K., Shimizu, B.
    • Journal Title

      Plant J. 55

      Pages: 989-999

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl-CoA by an 2-oxoglutarate dependent dioxygenase in Arabidopsis thaliana.2008

    • Author(s)
      Kai K, Mizutani M, Kawamura N. Yamamoto R, Tamai M, Yamaguchi H, Sakata K, Shimizu B
    • Journal Title

      Plant J. (In press)

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] アブシジン酸および植物ステロイドの代謝系に関わるシトクロムP450の酵素化学的研究2007

    • Author(s)
      水谷正治
    • Journal Title

      植物の生長調節 42

      Pages: 260-268

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] シトクロムP450の分子進化と植物の化学的多様性の獲得2007

    • Author(s)
      水谷正治
    • Journal Title

      蛋白質・核酸・酵素 52

      Pages: 1454-1464

    • NAID

      40015648125

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Annual Research Report 2007 Final Research Report Summary
  • [Journal Article] Biochemical stdies on cytochrome P450 monooxygenases in biosynthesis and catabolism of abscisic acid and plant steroids2007

    • Author(s)
      Mizutani, M
    • Journal Title

      Reg of Plant Growth Dev 42

      Pages: 19-29

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Correlation between molecular evolution of P450s and evolution of plant secondary metabolism2007

    • Author(s)
      Mizutani, M
    • Journal Title

      Tanpakushitsu Kakusan Koso 52

      Pages: 1454-64

    • NAID

      40015648125

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Cytochrome P450 CYP710A encodes the sterol C-22 desaturase in Arabidopsis and tomato.2006

    • Author(s)
      Morikawa T, Mizutani M, Aoki N, Watanabe B, Saga H, Saito S, Oikawa A, Suzuki H, Sakurai N, Shibata D, Wadano A, Sakata K, Ohta D.
    • Journal Title

      The Plant Cell 18

      Pages: 1008-1022

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] A plant growth retardant, uniconazole, is a potent inhibitor of ABA catabolism in Arabidopsis.2006

    • Author(s)
      Saito S, Okamoto M, Shinoda S, Kushiro T, Koshiba T, Kamiya Y, Hirai N, Todoroki Y, Sakata K, Nambara E, Mizutani M.
    • Journal Title

      Biosci Biotechnol Biochem 70

      Pages: 1713-1739

    • NAID

      10018529395

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] CYP724B2 and CYP90B3 function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.2006

    • Author(s)
      Ohnishi T, Watanabe B, Sakata K, Mizutani M.
    • Journal Title

      Biosci Biotechnol Biochem 70

      Pages: 2071-2080

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism.2006

    • Author(s)
      Ohnishi T, Nomura T, Watanabe B, Ohta D, Yokota T, Miyagawa H, Sakata K, Mizutani M.
    • Journal Title

      Phytochemistry 67

      Pages: 1895-1906

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Cytochrome P450 subfamily CYP710A genes encode sterol C-22 desaturase in plants.2006

    • Author(s)
      Morikawa T, Mizutani M, Ohta D.
    • Journal Title

      Biochem Soc Trans 34

      Pages: 1202-1205

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] C-23 hydroxylation by Arabidopsis CYP90C1 and CYP90D1 reveals a novel shortcut in brassinosteroid biosynthesis.2006

    • Author(s)
      Ohnishi T, Szatmari AM, Watanabe B, Fujita S, Bancos S, Koncz C, Lafos M, Shibata K, Yokota T, Sakata K, Szekeres M, Mizutani M.
    • Journal Title

      The Plant Cell 18

      Pages: 3275-3288

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Cytochrome P450 CYP710A encodes the sterol C-22 desaturase in Arabidopsis and tomato2006

    • Author(s)
      Morikawa T, Mizutani M, Aoki N, Watanabe B, Saga H, Saito S, Oikawa A, Suzuki H, Sakurai N, Shibata D, Wadano A, Sakata K, Ohta D.
    • Journal Title

      Plant Cell 18

      Pages: 1008-1022

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Uniconazole is a potent inhibitor of ABA 8'-hydroxylase in Arabidopsis.2006

    • Author(s)
      Saito, S., Okamoto, M, Shinoda, S., Kushiro, T., Koshiba T., Kamiya, Y., Hirai, N., Sakata, K., Nambara, E., Mizutani, M.
    • Journal Title

      Biosci. Biotechnol. Biochem. 70

      Pages: 1731-1739

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] CYP724B2 and CYP90B3 function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.2006

    • Author(s)
      Ohnishi, T., Watanabe, B., Sakata, K., Mizutani, M.
    • Journal Title

      Biosci. Biotechnol. Biochem. 70

      Pages: 2071-2080

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Tomato CYP734A7 catalyzes the C-26 hydroxylation of C_<27> and C_<28> brassinosteroids.2006

    • Author(s)
      Ohnishi, T., Nomura, T., Watanabe, B., Ohta, D., Yokota, T., Miyagawa, H., Sakata, K., Mizutani, M.
    • Journal Title

      Phytochemistry 67

      Pages: 1895-1906

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Cytochrome P450 subfamily CYP710A genes encodes sterol C-22 desaturase in plants.2006

    • Author(s)
      Morikawa, T., Mizutani, M., Ohta, D.
    • Journal Title

      Biochem Society Transact 34

      Pages: 1202-1205

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] C-23 hydroxylation by Arabidopsis CYP90C1 and CYP90D1 reveals a new shortcut route in brassinosteroid biosynthesis.2006

    • Author(s)
      Ohnishi, T., Bancos, S., Watanabe, B., Fujita, S., Szatmari, M., Koncz, C, Lafos, M., Yokota, T., Sakata, K., Szekeres, M., Mizutani, M
    • Journal Title

      Plant Cell 18

      Pages: 3275-3288

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] 大腸菌共発現系を用いたブラシノステロイドの酵素合成2008

    • Author(s)
      堀田順子、水谷正治
    • Organizer
      日本農芸化学会2008年度大会
    • Place of Presentation
      名城大学天白キャンパス
    • Year and Date
      2008-03-27
    • Related Report
      2007 Annual Research Report
  • [Presentation] アブシジン酸8'-水酸化酵素リガンドの基本構造2008

    • Author(s)
      上野琴巳、水谷正治、平井伸博、轟泰司
    • Organizer
      第49回日本植物生理学会年会
    • Place of Presentation
      札幌コンベンションセンター
    • Year and Date
      2008-03-22
    • Related Report
      2007 Annual Research Report

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Published: 2006-04-01   Modified: 2016-04-21  

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