真菌Fusicoccane型萜类环化酶催化机制系统性研究及其理性改造

Fusicoccane型萜类天然产物具有独特的5-8-5稠环骨架结构和多样的生物活性，引起了科学界的广泛兴趣。现报道该类化合物的数目已超过300，而已发现的该类环化酶却只有8个，包括文献报道的1个细菌和4个真菌来源及本课题组前期发现的3个真菌来源的酶，且绝大部分真菌酶催化的合环机制不清楚。此外，我们前期初步研究发现真菌酶与细菌酶在环化机制上有较大差别（ACS Catal. 2020, 10, 4306-4312）。基于前期工作，本项目拟运用量子化学计算、经典分子动力学模拟及QM/MM分子动力学模拟等理论手段和同位标记、定点突变等实验手段，对真菌中该类酶催化机制展开系统性研究，包括相关碳正离子内禀性、代表性酶的催化机理及酶的“监护”作用，总结酶调控相关规律，设计突变方案对酶的活性口袋进行理性改造以拓展这类酶的催化功能。本项目将解决真菌中该类酶催化过程中的诸多科学问题，具有多方面的研究意义。

Fusicoccane-type terpenoid natural products have a unique 5-8-5 fused-ring skeleton and diverse biological activities, which have attracted broad interest from the scientific community. It is reported that the number of such compounds has exceeded 300, while only eight such cyclases have been found, including one bacterial and four fungal sources reported by the literatures and three fungal sources previously discovered by our group. And most of the fungal enzyme-catalyzed the ring-closure mechanisms are unclear. Moreover, we found apparent differences between the catalytic mechanisms of fungal and bacterial enzymes in our early preliminary study (ACS Catal. 2020, 10, 4306 -4312). Based on the previous work, this project intends to use theoretical methods such as quantum chemical calculations, classical molecular dynamics simulations and QM/MM molecular dynamics simulations, as well as experimental methods such as isotope labeling and site-directed mutation, to carry out systematic studies on catalytic mechanism of such enzymes in fungi, including the intrinsic nature of the carbocations, the catalytic mechanisms of the representative enzymes and the "chaperone" role of the enzyme, summarize the relevant law of enzymatic regulation, and design mutant schemes to modify the active pockets of the enzymes, expanding the catalytic function of such enzymes. This project will solve many scientific problems in the catalytic process of this type of enzymes in fungi, and has multiple research significances.