适冷TfdB羟化多氯联苯类污染物催化机制及酶非专一性机制解析

Hydroxylase is catalytic initiation factor for metabolic pathway of many xenobiotic aromatic pollutants. Chlorophenols hydroxylase (TfdB-JLU) was cloned from polychlorinated biphenyls (PCBs) contaminated soil metagenome. This enzyme is cold-adapted and has complex substrates spectrum, and it has high potential for bioremediation use. In this study, on the basis of previous inspection of enzymatic hydroxylating PCBs degradation pathway and analysis of enzyme structure and promiscuity, we would like to use spectrum, HPLC-MS, NMR and other biophysical methods, to detect and analysis type and structure of the products of TfdB-JLU catalyzed PCBs substrates. Thermodynamic and kinetic studies are used to investigate energy changes as well as dynamics parameters of the conversion, and clarify the catalytic mechanism of enzyme hydroxylation degradation pathway. Directed evolution and rational design are used to screen and regulate enzyme promiscuity. Molecular docking and molecular dynamics methods are used to explore relationship of enzyme structure and hydroxylation function. Enzyme kinetic study as well as the foundation described above is used to reveal enzyme promiscuity mechanism during PCBs hydroxylation. The study is of great importance to clarify enzymatic bioremediation mechanism of aromatic pollutants, enrich and develop the enzyme promiscuity evolutionary theory and improve the practical application of enzyme bioremediation.

羟化酶是多种外源芳香污染物代谢途径的启动催化因子。我们对从多氯联苯（PCBs）污染土壤宏基因组中克隆的氯酚羟化酶(TfdB-JLU)的研究发现该酶兼具适冷和复杂底物谱的非专一特性，是极具潜力的生物修复用酶。本课题拟在前期对该酶羟化降解PCBs类底物途径预测和酶结构与非专一性分析的基础上，采用光谱、HPLC-MS和NMR等生物物理学手段，通过检测分析TfdB-JLU催化PCBs类底物反应产物种类和结构特征，运用酶热、动力学研究转化过程中体系能量和动力学等参数的变化及规律，解析该酶起始羟化降解途径的催化机制。通过定向进化和理性设计，筛选并调控TfdB-JLU对PCBs类底物的非专一性；采用分子对接和分子动力学等方法探讨该酶与PCBs类底物羟化作用的构效关系，结合酶动力学研究揭示其非专一性机制。该研究将对阐明芳香污染物酶促修复机制、丰富和发展酶非专一性进化理论和酶修复的实际应用等具有重要意义。

2,4-二氯苯酚羟化酶是2,4-D的生物降解过程的关键酶之一。本研究对TfdB-JLU羟化PCBs类污染物催化机制进行了解析，进一步确定了该酶为2,4-dichlorophenol 6-monooxygenase。通过对TfdB-JLU羟化PCBs非专一性的合理设计，分析了酶结构和非专一性的关系，通过对活性口袋及其内部关键氨基酸的调控，调控了酶的底物非专一性，总结了酶结构变化与非专一性变化之间的规律，初步建立了酶结构与非专一性之间的构效关系联系；加深了对酶结构与非专一性关系的理解，通过定点突变实验对酶非专一性进行有效调控，确定对酶非专一性有重要影响的氨基酸和区域。并结合分子生物学、生物信息学和酶动力学研究，基本阐明了酶底物非专一性的调控及分子识别机制。该研究将对阐明芳香污染物酶促修复机制、丰富和发展酶非专一性进化理论和酶修复的实际应用等具有重要意义。