Creation of non-natural enzyme reactions based on three-dimensional structure and expression screening
| Project/Area Number |
17H04763
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| Research Category |
Grant-in-Aid for Young Scientists (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biomolecular chemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
Awakawa Takayoshi 東京大学, 大学院薬学系研究科(薬学部), 准教授 (80609834)
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| Project Period (FY) |
2017-04-01 – 2021-03-31
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| Project Status |
Completed (Fiscal Year 2020)
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| Budget Amount *help |
¥16,640,000 (Direct Cost: ¥12,800,000、Indirect Cost: ¥3,840,000)
Fiscal Year 2020: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2019: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2018: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2017: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
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| Keywords | 生合成 / 酵素構造解析 / 物質生産 / 非天然型酵素反応 / テルペンインドール / 飛天年型酵素反応 / エンジニアリング / インドールテルペノイド / 二次代謝酵素 / 酵素立体構造 / 酵素精密機能解析 / 酵素機能改変 / 酸化酵素 / テルペンインドールアルカロイド / 生合成酵素 / X線結晶構造解析 / 反応解析 / 酵素反応 / 構造解析 |
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| Outline of Final Research Achievements |
Teleocidin B is a cyclic peptidic terpenoids characterized with 9-membered ring indolactam structure and a potent protein kinase C activator. In this study, we report the mechanistic characterization of indolactam-forming P450 enzymes TleB and its homolog HinD. Interestingly, the TleB and HinD accepted the various dipeptide substrates, and generated not only indolactam analogs but also unusual 6/5/6 ring structures in vitro. Comparisons of the complex crystal structures with different substrates revealed the important residues and possible binding modes in the active site. Combination of the findings from the enzyme assays and X-ray crystal structures of TleB and HinD, we proposed a mechanism of the enzyme catalyzed intramolecular C(sp2)-H bonds amination. We also identified the reaction of HinD homologs from Streptoalloteichus hindustanus.
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| Academic Significance and Societal Importance of the Research Achievements |
本研究では、環境に優しく、立体構造の形成に秀でた生合成酵素を改変し、非天然型の基質を作用させることで、多数の新規化合物を取得した。テルペンインドール化合物テレオシジン生合成酵素のうち特異なP450酸化酵素TleBを例として、X線結晶構造解析を行い、その立体構造を明らかにし、その触媒メカニズムを明らかにした。本結果は、酵素を用いた物質生産系構築のための基盤を構築し、多様な代謝経路で重要な役割を果たすP450酸化酵素の新たな触媒能力を解明することで、幅広い学術領域に大きなインパクトを与えた。これを基盤に、新たな物質生産の方法論が提唱され、医薬品製造技術への貢献を通して、公共の福祉に大きく貢献する。
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Report
(5 results)
Research Products
(72 results)
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[Journal Article] Biosynthetic pathway for furanosteroid demethoxyviridin and identification of an unusual pregnane side-chain cleavage2018
Author(s)
2.Wang, G.-Q., Chen, G.-D., Qin, S.-Y., Hu, D., Awakawa, T., Li, S.-Y., Lv, J.-M., Wang, C.-X., Yao, X.-S., Abe, I., Gao, H.
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Journal Title
Nature Communications
Volume: 9
Issue: 1
Pages: 1838-1838
DOI
Related Report
Peer Reviewed / Open Access / Int'l Joint Research
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