倍半萜合酶FSTS/SDS催化机制及其化学多样性调控研究

Natural product sesquiterpenes are abundant in number and diverse in structure, providing a wealth of resources for drug design. Because of the complex molecular chirality and multiple condensed rings, chemical synthesis of sesquiterpenes and their derivatives is often challenging; while the biosynthesis has advantages of short cycle and high efficiency, and has important application prospect in drug discovery. Deciphering the enzyme-catalyzed mechanisms and the chemo-/stereo-selective regulatory factors of sesquiterpenes can provide important theoretical guidance for the rational design of sesquiterpenoid biosynthesis. Our previous work has preliminarily revealed the correspondence between substrate folding and chemo-/stereo-selectivity in FSTS sesquiterpene synthase (ACS Catal. 2018, 8, 1363; ACS Catal. 2017, 7, 5841). This project will continue the previous work to conduct a more in-depth study on the catalytic mechanisms of 1,6-ring closure FSTS and 1,10-ring closure SDS sesquiterpene synthase in order to resolve other relevant factors for enzymatic regulation. By comparing the related regulatory of chemo-/stereo-selectivity factors between FSTS and SDS, we hope to find out the differences between the 1,6- and 1,10-cyclization pathway catalyzed by the enzyme, and summarize the universal rules related to enzyme catalysis regulation. Finally, according to the summarized rules, the reforming of active pocket of enzyme will be theoretically simulated to predict the chemical diversity of products and intermediates of enzyme catalysis, which will shed light on the rational design of the sesquiterpenoid biosynthesis.

倍半萜类天然产物数量庞大、结构多样。因其复杂的分子手性及多种稠环方式，倍半萜及其衍生物的化学合成往往充满挑战，而生物合成则有周期短、效率高的优势，在药物开发中有着重要的应用前景。解析其酶催化机制及化学、立体选择性调控因素，能为倍半萜生物合成的理性设计提供重要的理论指导。前期工作(ACS Catal 2017/2018)已初步揭示了FSTS倍半萜合酶中底物折叠与化学选择性的对应关系。本项目将进一步对1,6-合环FSTS和1,10-合环SDS倍半萜合酶的催化机制进行更为深入的研究，解析酶催化调控的其他相关因素；通过比较分析FSTS和SDS酶催化化学与立体选择性的调控因素，探寻调控倍半萜合酶催化1,6-和1,10-合环反应的普适性规律；最后，基于酶催化机制及其调控规律，改造酶活性口袋并进行理论模拟预测其酶催化产物、中间体的化学多样性，为倍半萜合酶生物合成的理性设计指明方向。

萜类合成酶催化自然界最为复杂的环化串联反应，为自然界最大的一类天然产物提供多样的骨架分子。这些环化串联改变了一半以上底物碳原子的成键及杂化方式，同时引入多个手性中心，而所有的这些转化都在一步酶促反应中完成。萜类化合物的多样性源于生物反应过程中碳正离子中间体形态的多样性，而倍半萜碳正离子中间体的形态最为多样。解析其酶催化机制及化学、立体选择性调控因素，能为倍半萜生物合成的理性设计提供重要的理论指导。本项目选取代表性的倍半萜环化酶，展开酶催化机制研究，并解析影响碳正离子中间体形态的关键因素。利用QM/MM分子动力学模拟获得了实验上无法获得的酶催化动态结构数据及反应过程中的能量数据。在三个方面取得了重要进展：（1）解决了该领域长期未找到答案的一大机制难题⸺酶是如何直接干预去质子化-再质子化序列的，打开了研究萜类生物合成中去质子化-再质子化序列催化细节的一扇大门，为其它萜类环化酶的研究指明了方向；（2）剖析了酶的区域/立体化学调控机制，对萜类环化酶的理性改造具有指导意义；（3）在区域/立体化学调控理论的指导下成功实现了对环化酶MgMS的初步理性改造，对后续研究具有借鉴意义。本项目部分研究成果已发表高水平SCI论文4篇，其中影响因子大于10的论文4篇，大于40的论文1篇。