碳-氢键区域和立体选择性多样化P450过加氧酶体系的构筑及其分子机理研究

C-H键的区域和立体选择多样性羟化能实现底物多个不同位点的功能化修饰，对广泛碳氢化合物的高附加值综合利用具有重要意义。但目前策略研究还未实现同一酶工程体系对多个C-H键选择多样性羟化的良好调控。本项目拟在双功能小分子协同P450催化策略基础上，以P450酶催化烷基苯羟化为模式反应进行研究，通过酶的半理性设计与双功能小分子的非共价动态修饰和化学干预作用双重调控有机结合，精细调节底物空腔，实现多个C-H键的特异识别羟化。首先，选取活性中心关键位点进行半理性设计组合突变筛选，然后结合小分子结构优化调控，产生不同位点C-H键的选择多样性；后续通过底物空腔周围特定氨基酸位点的定点突变及迭代突变协同小分子结构微调，实现烷基苯多个C-H键的高效选择多样性羟化；最后利用晶体学和分子动力学模拟来阐明实现不同C-H键选择的分子机理，为构建人工C-H键选择多样性羟化P450调控体系提供理论基础和技术支撑。

Regiodivergent and stereodivergent hydroxylation of multiple C-H bonds can functionally modify different sites of one substrate, which is of great significance for comprehensive utilization of a wide range of hydrocarbons with high added value. However, the current strategies have not been able to achieve a good control to selective divergent hydroxylation of multiple C-H bonds by one enzyme system. On the basis of the Dual-functional small molecule(DFSM) facilitated P450 peroxygenase strategy, we intends to study the hydroxylation of alkylbenzene as a model reaction. Through the combination of semi-rational design and the non-covalent dynamic chemical modification and chemical intervention of rationally designed DFSMs, adjust substrate cavity finely and achieve the selective divergent C-H hydroxylation of alkylbenzene catalyzed by one P450 peroxygenase system. Firstly, screen the combinatorial mutants of the selected key sites close the active center by semi-rational design, then combine the structural control of DFSMs to generate the divergent selectivity of different sites. Further combine site-directed mutation and iterative mutation of key amino acid sites around the substrate cavity with fine-tuning of DFSMs to achieve selective divergent hydroxylation of multiple C-H bonds of alkylbenzene efficiently. Then elucidate the molecular mechanisms of divergent selectivity by crystallography and molecular dynamics simulations. This study will provide theoretical basis and technical support for developing the artificial regiodivergent and stereodivergent C-H hydroxylation P450 system.