聚焦铰链结构的酶构象动力学研究与理性改造

构象动力学改造是增强酶蛋白催化性能或使酶进化获得新催化功能的前沿技术。但是，由于发展时间较短，尚缺乏合适、明确的改造热点，极大限制了其广泛应用。前期研究表明，“铰链弯曲运动”酶的铰链结构是一个极具潜力的构象动力学改造热点。如能建立聚焦铰链结构的构象动力学理性改造策略，有助于开发催化性能更强的酶蛋白，并且能丰富酶结构-构象-功能关系的理论内涵，具有重要的现实意义与学术价值。然而，铰链结构变化影响酶构象动力学与催化活力的分子机制尚未解析，针对铰链进行理性设计以精准调控酶构象动力学的具体方法也有待建立。本项目将以氨基酸脱氢酶作为模型蛋白，借助铰链聚焦定向进化、分子动力学模拟等手段系统考察铰链结构变化影响酶构象动力学与催化活力的方式及潜在规律；进一步探索通过铰链结构理性改造调控酶构象动力学、定向提高酶蛋白催化活力，建立铰链聚焦的构象动力学理性改造策略，为高性能酶催化剂的构建提供新思路和新方法。

Conformational dynamic engineering is a cutting-edge technology for enhancing the catalytic performance of enzymes or evolving enzymes to obtain new catalytic functions. However, due to the short development time, there is still a lack of suitable and well-studied hot spots for conformational dynamic engineering, which greatly limits its extensive application. The previous research has shown that the hinge structure of hinge-bending enzymes represents a potential hotspot for conformational dynamic engineering. Establishing the hinge-focused rational conformational dynamics engineering strategy would aid the development of high-performance biocatalysts, and enhance our understanding of structure-conformation-function relationships of enzymes, thus has both practical and scientific value. However, the molecular mechanism of how hinge structure mutation affecting enzyme conformational dynamics and catalytic activity has not yet been resolved, and specific protocols for rational design the hinge structure to precisely modulate enzyme conformational dynamics has not been established. In our proposal, using amino acid dehydrogenases (AADHs) as a model, we will apply hinge-focused directed evolution, molecular dynamics simulation and other methods to systematically investigate how hinge structure changes affecting enzymes’ conformational dynamics and catalytic function. Next, we would design the hinge structure rationally to tune enzymes’ conformational dynamics to improve their catalytic activity, and then establish the hinge-focused rational conformational dynamics engineering strategy, providing new insights and new methods for the high-performance enzymes engineering.