Structural Basis for Selectivity in Flavin-Dependent Monooxygenase-Catalyzed Oxidative Dearomatization
Structural Basis for Selectivity in Flavin-Dependent Monooxygenase-Catalyzed Oxidative Dearomatization
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DOI：
10.1021/acscatal.8b04575
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发表时间：
2019-04-01
期刊：
影响因子：
12.9
通讯作者：
Narayan, Alison R. H.
中科院分区：
文献类型：
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作者：
Benitez, Attabey Rodriguez;Tweedy, Sara E.;Narayan, Alison R. H.
生物催化反应体现了理想化学转化的许多特征，包括无可挑剔的选择性，高催化效率，温和的反应条件和使用环境友好的试剂。这些优势创造了对生物催化剂的需求，这些生物催化剂扩大了合成化学家可用的复杂生成反应的组合。然而，在生物催化剂的底物范围和其选择性之间经常存在的权衡限制了酶在合成中的应用。我们最近证明，黄素依赖性单加氧酶，TropB，保持高水平的网站和立体选择性在一系列结构不同的基板。在本文中，我们公开了TropB中底物结合的结构基础，其在一系列酚底物中进行具有精致位点和立体选择性的合成挑战性不对称氧化脱芳构化反应，为未来的蛋白质工程和反应开发工作提供了基础。我们的假设基板结合的TropB和分子动力学模拟与相应的计算TropB模型的晶体结构的通知，并支持实验数据。与典型的A类FAD依赖性单加氧酶相比，其中底物以质子化形式结合，我们的数据表明底物的酚盐形式结合在活性位点中。此外，底物的位置是通过两点结合的酚氧Arg 206和Tyr 239，这被证明有不同的和必不可少的作用，在催化控制。Arg 206参与黄素辅因子的减少，表明在黄素动力学中的作用。此外，QM/MM模拟揭示的相互作用，管理的面部选择性，导致一个高度对映选择性的转换。因此，阐明了TropB在一系列底物反应中观察到的高水平位点和立体选择性的结构起源，为未来的蛋白质工程和反应开发工作提供了基础。
Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions, and the use of environmentally benign reagents. These advantages have created a demand for biocatalysts that expand the portfolio of complexity-generating reactions available to synthetic chemists. However, the trade-off that often exists between the substrate scope of a biocatalyst and its selectivity limits the application of enzymes in synthesis. We recently demonstrated that a flavin-dependent monooxygenase, TropB, maintains high levels of site-and stereoselectivity across a range of structurally diverse substrates. Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site- and stereoselectivity across a range of phenol substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by a crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data. In contrast to canonical class A FAD-dependent monooxygenases in which substrates bind in a protonated form, our data indicate that the phenolate form of the substrate binds in the active site. Furthermore, the substrate position is controlled through two-point binding of the phenolate oxygen to Arg206 and Tyr239, which are shown to have distinct and essential roles in catalysis. Arg206 is involved in the reduction of the flavin cofactor, suggesting a role in flavin dynamics. Further, QM/MM simulations reveal the interactions that govern the facial selectivity that leads to a highly enantioselective transformation. Thus, the structural origins of the high levels of site- and stereoselectivity observed in reactions of TropB across a range of substrates are elucidated, providing a foundation for future protein engineering and reaction development efforts.