基因融合影响腈水合酶活性和稳定性的作用机制解析

腈水合酶(NHase)是含有α和β两个亚基的金属酶。我们采用基因融合策略，通过在NHase的α和β亚基中间添加一个连接肽(linker)，得到了一种高活性和高稳定性的亚基融合NHase。基因融合是一种提高蛋白质性能的有效策略，然而并不是所有的基因融合都可以提高蛋白质性能。本研究旨在阐明亚基融合模式提高NHase活性和稳定性的生理机制。选用长短及性质不同的linker融合NHase的两个亚基，筛选一系列活性和稳定性有一定差异的融合酶；通过同源建模、分子动力学及拉伸动力学模拟，比较原始酶和不同融合酶的分子结构，明确影响融合酶活性和稳定性的关键因素，并阐明这些因素影响酶活性和稳定性作用机制。在此基础上，结合融合NHase连接端的结构特点，归纳不同linker影响NHase活性和稳定性的规律，为基于基因融合的蛋白质定向改造提供理论依据。

Nitrile hydratase (NHase) is one of the metalloenzymes comprised of α and β subunits. We successfully constructed a subunit-fused NHase exhibiting high catalytic activity and stability by fusing the β- and α-subunits based on gene fusion strategy. The improvement of fusion NHase’s activity and stability should be ascribed to the protein structure transformation after subunit fusion. Gene fusion can enhance protein stability, however, not all protein stability can be enhanced by gene fusion. This study aims to clarify such relevance. We try to obtain various fused enzyme with distinct activity or stability by linker substitution inside fusion NHase. To explore the reason why these different fusion NHases exhibited diverse activity or stability, molecular modeling, molecular dynamics simulation as well as steered molecular dynamics simulation are carried out. We try to select the key factors effecting the activity and stability and clarify the mechanisms. Moreover, we try to summarize the rules of the relationship between linker and the function of fused-NHases based on the analysis the structure of linking terminal of NHases. This study shed light on the directed evolution of subunit-fused proteins.