Mycobacterium vanbaalenii PYR-1多环芳烃双加氧酶的结构与催化功能的研究

多环芳烃含有两个及以上聚集的苯环，其中16种被列优先污染物。目前已分离到多株多环芳烃降解菌，但仅两环的萘的降解起始双加氧酶的结构与催化功能得到解析与研究。高毒性的三环的蒽和菲、四环的芘和荧蒽（均为优先污染物）等高环多环芳烃降解的关键酶在酶学和结构水平均未验证。特别是高环多环芳烃双加氧酶尚未获得有催化活性的纯酶。本项目拟以多环芳烃模式降解菌Mycobacterium vanbaalenii PYR-1中的高环多环芳烃多组份双加氧酶为研究对象，无氧和有氧条件相结合纯化该酶各组分并获得晶体；以菲、蒽、芘和荧蒽为底物，检测纯酶催化活性、酶动力学参数等；解析酶结构，确认聚合形式、底物通道、活性位点等；通过分子动力学模拟解释底物偏好。在结构和生化水平阐明高环多环芳烃双加氧酶的催化功能及结构与功能的关系。揭示高环多环芳烃双加氧酶的适应性进化机制，为选择性高效降解高环多环芳烃及其污染环境治理提供理论基础。

Polycyclic aromatic hydrocarbons (PAHs) are a class of aromatic compounds composed of two or more fused benzene rings. As many as 16 PAHs have been classified as priority pollutants. So far, a number of PAHs utilizers including Mycobacterium vanbaalenii PYR-1 have been isolated. But only the structure and catalytic functions of the initial dioxygenase involved in naphthalene (with two benzene rings) degradation have been resolved. The dioxygenases involved in the initial degradation of more toxic high-molecular-weight PAHs including anthracene, phenanthrene, pyrene and fluoranthene (all are priority pollutants), have not been verified at the enzymatic and structural levels. No purified high-molecular-weight PAHs dioxygenase with catalytic activity has been reported. This project will study the structure and function of a newly defined active high-molecular-weight PAHs dioxygenase system FNidA3B3-PhtAcAd from strain PYR-1, using high-molecular-weight PAHs as substrates, including anthracene, phenanthrene, fluoranthene and pyrene. The different components of the aforementioned dioxygenase will be purified under anaerobic and aerobic conditions, and the core component crystals will be obtained under anaerobic conditions. The crystal structure of the dioxygenase will be resolved at a high resolution. This will help us to analyze the polymerization form, the substrate channel, active sites of this enzyme. Its substrate specificity and preference as well as its kinetic parameters will also be examined by conventional assays and molecular dynamics simulation. Eventually, the catalytic function and the crystal structure of this high-molecular-weight PAHs dioxygenase will be elucidated at the molecular, biochemical and structural levels. This study will not only provide us a better understanding of the adaptive evolutionary mechanisms of high-molecular-weight PAHs dioxygenases, but also create a theoretical basis for the further development of the treatment of PAHs polluted environments via synthetic biology.