含氮杂环吡啶环化合物是重要的环境污染物和环境毒物。由于环上氮原子的吸电子作用，吡啶环较稳定，不易被氧化。吡啶环β位单加氧酶在微生物降解这些化合物的途径中起到活化吡啶环的重要作用，便于双加氧酶将其开环断裂。我们拟研究含吡啶环化合物的微生物分解代谢途径中3个代表性的吡啶环β位单加氧酶：假单胞菌S16菌株中的6-羟基-3-琥珀酰吡啶单加氧酶HspB及其同工酶HspA、KT2440菌株中的6-羟基烟酸单加氧酶NicC，解析它们自身及与各自底物的复合物的结构，考察它们结构上的共同性，并揭示它们识别吡啶环底物的分子机理；通过计算生物学研究，阐明它们专一性催化吡啶环β位羟基化的反应机制；通过突变体酶活性检测和微生物代谢功能分析，验证吡啶环β位单加氧酶的关键残基对酶活性和代谢功能的重要作用。本项目将加深对微生物降解环境污染物途径中的吡啶环β位单加氧酶的认识，为利用微生物技术清除含氮杂环污染物奠定理论基础。

Pyridine-containing heterocyclic compounds are one major kind of environmental pollutants of our country. Because of the electron withdrawing effect of the nitrogen atom on the pyridine ring, pyridine rings are more stable than benzene rings and are more difficult to be oxidized. Pyridine ring β-monooxygenases play important roles in microbial degradation pathways for pyridine-containing compounds. They activate pyridine rings by modifying them with hydroxyl groups at β- or α- positions, to make it easier for subsequent dioxygenases to oxidatively cleave the pyridine rings. In this proposal, we plan to study three microbial pyridine ring β-monooxygenases: 6-hydroxyl-3-succinoyl-pyridine (HSP) monooxygenases HspB and HspA in the nicotine degradation pathway of Pseudomonas putida S16, and 6-hydroxyl-nicotinic acid monooxygenase NicC in the nicotinic acid degradation pathway of Pseudomonas putida KT2440. For example, in the HspB-catalyzed reaction, there is one co-factor (FAD) and four substrates (HSP, NADH, dioxygen, and water) involved, and four products (2,5-dihydroxypyridine, NAD+, succinate, and water) are formed. In the reactions catalyzed by HspA and NicC, FAD and NADH are also involved. We are going to determine the crystal structures of these pyridine ring β-monooxygenases by themselves as well as the crystal structures of these monooxygenases in complexes with their respective pyridine ring-containing substrates. By examining these structures as well as carrying out computer molecular dynamics simulation and quantum chemical calculation, we are going to study how these monooxygenases specifically recognize their respective pyridine ring-containing substrates, and selectively catalyze the hydroxylation reactions at β-positions of pyridine rings. By means of structural analysis, we are going to identify key residues of these monooxygenases which play critical roles in recognizing substrates and catalyzing β-hydroxylation reactions on pyridine rings. We are going to perform enzymatic activity assays and microbial functional assays using site-directed mutants to elucidate which residues are essential for the catalytic activities of these monooxygenases and for the metabolic capabilities of microbes towards their respective pyridine ring-containing compounds. Through studying of these three representative enzymes, we are going to investigate important properties of pyridine ring β-monooxygenases in microbial degradation pathways of pyridine ring-containing environmental pollutants.