Benítez Attabey Rodríguez, Tweedy Sara, Baker Dockrey Summer A, Lukowski April L, Wymore Troy, Khare Dheeraj, Brooks Charles L, Palfey Bruce A, Smith Janet L, Narayan Alison R H
Program in Chemical Biology, University of Michigan, Ann arbor Michigan 48109.
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
ACS Catal. 2019 Apr 5;9(4):3633-3640. doi: 10.1021/acscatal.8b04575. Epub 2019 Mar 25.
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 tradeoff 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 twopoint 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.
生物催化反应体现了理想化学转化的许多特征，包括具有无可挑剔的选择性、高催化效率、温和的反应条件以及使用环境友好型试剂的潜力。这些优势催生了对生物催化剂的需求，这类催化剂能够拓展合成化学家可利用的生成复杂性反应的种类。然而，生物催化剂的底物范围与其选择性之间通常存在的权衡限制了酶在合成中的应用。我们最近证明，一种黄素依赖性单加氧酶TropB在一系列结构多样的底物上都能保持高水平的位点选择性和立体选择性。在此，我们揭示了TropB中底物结合的结构基础，TropB能在一系列酚类底物上以极高的位点选择性和立体选择性进行具有合成挑战性的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奠定了基础。我们关于底物结合的假设是基于TropB的晶体结构以及与相应的计算TropB模型进行的分子动力学模拟得出的，并得到了实验数据的支持。与典型的A类黄素依赖性单加氧酶不同，在后者中底物以质子化形式结合，而我们的数据表明底物的酚盐形式结合在活性位点。此外，底物的位置是通过酚盐氧与Arg206和Tyr239的两点结合来控制的，这两个氨基酸在催化过程中具有不同且至关重要的作用。Arg206参与黄素辅因子的还原，表明其在黄素动力学中发挥作用。此外，量子力学/分子力学模拟揭示了决定面选择性从而导致高度对映选择性转化的相互作用。因此，阐明了在TropB与一系列底物的反应中观察到的高水平位点选择性和立体选择性的结构根源，为未来的蛋白质工程和反应开发工作提供了基础。
