開いた金属の調整部位に隣接する調節可能な基板結合ポケットを持つヘム酵素の設計
Indrek Kalvet1,2,3, Mary Ortmayer4, Jingming Zhao4
1Institute for Protein Design, University of Washington, 3946 W Stevens Way NE, Seattle, Washington 98195, United States.
Journal of the American Chemical Society
|June 21, 2023
まとめ
科学者は新しいヘム結合タンパク質 dnHEM1 を設計し,シトクロームP450sを模倣した. このタンパク質構造は,プロフェンスの過酸化とエナチオセレクティブのカーベノトランスファーゼ機能を含む多様な触媒活動のためのカスタム酵素設計を可能にします.
科学分野:
- 生物化学
- タンパク質工学
- 酵素触媒
背景:
- サイトクロムP450のようなヘム酵素は,鉄-ポルフィリン共因子と開いた基板結合ポケットにより,驚くべき触媒的多用性を示す.
- 以前のヘム結合タンパク質の設計には,P450のような基板ポケットが欠け,その触媒作用範囲が制限されていた.
研究 の 目的:
- P450の触媒部位の幾何学とカスタムタンパク質の設計の柔軟性を組み合わせた新しいタンパク質の構造 (dnHEM1) を設計する.
- 基質相互作用と反応性中間生成のための調整可能な遠端ポケットを持つ高親和性ヘム結合タンパク質を作成する.
主な方法:
- 軸性ヒスティジンリガンドとヘムコファクターを結合させる螺旋束のエスカフォードのデノボタンパク質設計.
- 設計されたタンパク質 (dnHEM1) の構造を1.6 Åの解像度で決定するためのX線結晶学.
- ペロキシダゼとカルベノトランスファーゼの活性化のために,遠端のポケットを改造することによって,タンパク質工学.
主要な成果:
- dnHEM1の結晶構造は,プログラムされた重要な要素を備えた設計モデルと優れた一致を証明しました.
- エンジニアリングされたdnhem1の変種は,安定した中性フェリル中間体で熟練した過酸化活性を示した.
- 再設計されたdnHEM1は,高度にエナチオセレクティブなスタイレン・サイクロプロパネーション (最大93%の収量,97:3 e.r.) を達成した. カーベノトランスファーゼの活性によって
結論:
- 開発された de novo タンパク質設計アプローチは,カスタム化されたコファクターと結合ポケットを持つ酵素の作成を可能にします.
- この戦略は酵素機能の精密な設計を可能にし,生物触媒と合成化学の可能性を広げます.
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