2007 Fiscal Year Final Research Report Summary
Construction of plant oxygenase libraries and their functional characterization
| Project/Area Number |
18580091
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Kyoto University |
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Principal Investigator |
MIZUTANI Masaharu Kyoto University, Institute for Chemical Research, Assistant Professor (60303898)
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| Project Period (FY) |
2006 – 2007
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| Keywords | Cytochrome P450 / Flavin monooxygenase / 2-oxoglutarate dependent dioxygenase / Oxygenase / Arabidopsis |
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| 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
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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
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[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.
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Journal Title
Plant J. 55
Pages: 989-999
Description
「研究成果報告書概要(欧文)」より
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[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.
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Journal Title
The Plant Cell 18
Pages: 1008-1022
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[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.
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Journal Title
Biosci Biotechnol Biochem 70
Pages: 1713-1739
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[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.
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Journal Title
The Plant Cell 18
Pages: 3275-3288
Description
「研究成果報告書概要(和文)」より
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[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.
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Journal Title
Plant Cell 18
Pages: 1008-1022
Description
「研究成果報告書概要(欧文)」より
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[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.
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Journal Title
Biosci. Biotechnol. Biochem. 70
Pages: 1731-1739
Description
「研究成果報告書概要(欧文)」より
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[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.
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Journal Title
Phytochemistry 67
Pages: 1895-1906
Description
「研究成果報告書概要(欧文)」より
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[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
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Journal Title
Plant Cell 18
Pages: 3275-3288
Description
「研究成果報告書概要(欧文)」より
