2021 Fiscal Year Final Research Report
Integrating Computational and Experimental Approaches for New Paradigms in Molecule Synthesis
| Project/Area Number |
19KK0150
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| Research Category |
Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
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| Allocation Type | Multi-year Fund |
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| Review Section |
Medium-sized Section 38:Agricultural chemistry and related fields
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| Research Institution | University of Shizuoka |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
佐藤 道大 静岡県立大学, 薬学部, 講師 (10629695)
恒松 雄太 静岡県立大学, 薬学部, 講師 (30629697)
岸本 真治 静岡県立大学, 薬学部, 助教 (40814330)
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| Project Period (FY) |
2019-10-07 – 2022-03-31
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| Keywords | 天然物 / 生合成 / Diels-Alder / 反応メカニズム |
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| Outline of Final Research Achievements |
Here we show the crystal structure of CghA–product complex at 2.0 resolution. Our result provides the second structural information of eukaryotic Diels–Alderases and adds yet another fold to the family of proteins reported to catalyse [4+2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured exon adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels-Alder reactions, and paves the way toward developing enzymes potentially useful in various bio/synthetic applications.
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| Free Research Field |
天然物生合成
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| Academic Significance and Societal Importance of the Research Achievements |
リポカリン型の構造を有するDAaseは報告がなく, 天然におけるDAaseの収斂進化を示唆するものであった。またCghA-1の複合体結晶構造解析および変異体の速度論解析からは, 酵素の基質許容に関する知見が得られ, ジエノフィルの電子求引に関わるアミノ酸残基を明らかにすることができた。さらに立体選択性に関しては, 基質のコンフォメーションをendoではなくexo付加に優位となるよう改変することで, 立体選択性の異なる酵素の創製に成功した。また, DAaseの生成物阻害回避機構については, 計算解析による遷移状態の自由エネルギーを算出することで, 合理的な説明が可能となった。
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