Department of Chemistry and Institute for Nanotechnology & Advanced Materials, Bar-Ilan University, Ramat-Gan 52900, Israel.
Institut für Chemie und Biochemie, Strukturbiochemie, Freie Universität Berlin, Takustr. 6, 14195 Berlin, Germany.
J Am Chem Soc. 2020 Dec 23;142(51):21562-21574. doi: 10.1021/jacs.0c11348. Epub 2020 Dec 8.
Terpene synthases generate terpenes employing diversified carbocation chemistry, including highly specific ring formations, proton and hydride transfers, and methyl as well as methylene migrations, followed by reaction quenching. In this enzyme family, the main catalytic challenge is not rate enhancement, but rather structural and reactive control of intrinsically unstable carbocations in order to guide the resulting product distribution. Here we employ multiscale modeling within classical and quantum dynamics frameworks to investigate the reaction mechanism in the diterpene synthase CotB2, commencing with the substrate geranyl geranyl diphosphate and terminating with the carbocation precursor to the final product cyclooctat-9-en-7-ol. The 11-step in-enzyme carbocation cascade is compared with the same reaction in the absence of the enzyme. Remarkably, the free energy profiles in gas phase and in CotB2 are surprisingly similar. This similarity contrasts the multitude of strong π-cation, dipole-cation, and ion-pair interactions between all intermediates in the reaction cascade and the enzyme, suggesting a remarkable balance of interactions in CotB2. We ascribe this balance to the similar magnitude of the interactions between the carbocations along the reaction coordinate and the enzyme environment. The effect of CotB2 mutations is studied using multiscale mechanistic docking, machine learning, and X-ray crystallography, pointing the way for future terpene synthase design.
萜烯合酶利用多样化的碳正离子化学生成萜烯，包括高度特异性的环形成、质子和氢转移以及甲基和亚甲基迁移，然后进行反应淬灭。在这个酶家族中，主要的催化挑战不是提高速率，而是对内在不稳定的碳正离子进行结构和反应控制，以引导产生的产物分布。在这里，我们采用经典和量子动力学框架内的多尺度建模来研究二萜合酶 CotB2 中的反应机制，从底物香叶基香叶基二磷酸开始，以最终产物环辛-9-烯-7-醇的碳正离子前体结束。酶内的 11 步碳正离子级联与没有酶的相同反应进行了比较。值得注意的是，气相中和 CotB2 中的自由能曲线非常相似。这种相似性与反应级联中的所有中间体与酶之间存在的大量强π-阳离子、偶极-阳离子和离子对相互作用形成鲜明对比，表明 CotB2 中存在着显著的相互作用平衡。我们将这种平衡归因于沿反应坐标的碳正离子和酶环境之间相互作用的大小相似。使用多尺度机械对接、机器学习和 X 射线晶体学研究 CotB2 突变的影响，为未来的萜烯合酶设计指明了方向。
