Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C-H Functionalization and Epoxidation of Tirandamycin Antibiotics.
Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C-H Functionalization and Epoxidation of Tirandamycin Antibiotics.
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DOI：
10.1021/acscatal.1c01460
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发表时间：
2021-07-02
期刊：
影响因子：
12.9
通讯作者：
Sherman DH
中科院分区：
文献类型：
--
作者：
Espinoza RV;Haatveit KC;Grossman SW;Tan JY;McGlade CA;Khatri Y;Newmister SA;Schmidt JJ;Garcia-Borràs M;Montgomery J;Houk KN;Sherman DH
迭代P450酶是用于复杂天然产物支架的选择性后期C-H氧化的强大生物催化剂。这些酶代表了选择性和级联反应的有用工具，有助于直接获得核心结构多样化。最近，我们报道了多功能细菌P450 TamI的结构，并阐明了其底物结合的分子基础和在底物的不同碳原子上的严格反应顺序。在这里，我们报告了TamI生物催化剂工具箱的设计和表征，该工具箱由Leu 101、Leu 244和/或Leu 295突变产生，这些突变会改变天然选择性、步骤顺序和催化反应的数量，包括工程改造能够催化的变体。在没有黄素蛋白和氧化伴侣TamL的帮助下进行四步氧化级联反应。调谐的酶超越固有的底物反应性，使催化剂控制的C-H官能化和烯烃环氧化的含特特拉姆酸的天然产物替兰达霉素。通过TamI介导的酶促合成产生五种生物活性的替兰达霉素衍生物（6-10）。量子力学计算和MD模拟提供了重要的见解的基础上改变的选择性和潜在的生物催化机制，增强连续氧化的迭代P450 TamI。
Iterative P450 enzymes are powerful biocatalysts for selective late-stage C–H oxidation of complex natural product scaffolds. These enzymes represent useful tools for selectivity and cascade reactions, facilitating direct access to core structure diversification. Recently, we reported the structure of the multifunctional bacterial P450 TamI and elucidated the molecular basis of its substrate binding and strict reaction sequence at distinct carbon atoms of the substrate. Here, we report the design and characterization of a toolbox of TamI biocatalysts, generated by mutations at Leu101, Leu244, and/or Leu295, that alter the native selectivity, step sequence, and number of reactions catalyzed, including the engineering of a variant capable of catalyzing a four-step oxidative cascade without the assistance of the flavoprotein and oxidative partner TamL. The tuned enzymes override inherent substrate reactivity, enabling catalyst-controlled C–H functionalization and alkene epoxidation of the tetramic acid-containing natural product tirandamycin. Five bioactive tirandamycin derivatives (6–10) were generated through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD simulations provide important insights into the basis of altered selectivity and underlying biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI.
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影响因子：
12.9
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通讯作者：
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