Creation of non-natural enzyme reactions based on three-dimensional structure and expression screening

2020 Fiscal Year Final Research Report

• Project/Area Number: 17H04763
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Single-year Grants
• Research Field: Biomolecular chemistry
• Research Institution: The University of Tokyo
• Principal Investigator: Awakawa Takayoshi 東京大学, 大学院薬学系研究科(薬学部), 准教授 (80609834)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Keywords: 生合成 / 酵素構造解析 / 物質生産

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.

Free Research Field

天然物化学、酵素工学、合成生物学

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。