微生物降解含氮杂环途径中的吡啶环β位单加氧酶的结构和功能分析

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.

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。

含氮杂环芳香化合物（N-heterocyclic aromatic compounds，NHACs）是工业溶剂、染料、药物以及杀虫剂的重要成分，通过人类活动进入环境，给地球上的地下水和土壤带来严重的污染。研究表明，含氮杂环芳香化合物对有机体有诱导突变甚至致癌的风险，因此寻找有效的途径降解环境中的含氮杂环化合物，对于维护人类健康及保护生态环境具有重要意义。近年来研究发现，含氮杂环芳香化合物通过生物酶（羟基化酶/单加氧酶/氧化还原酶）的氧化还原过程降解成无害甚至有用的分子。我们主要研究了在微生物对含氮杂环芳香化合物的降解途径中发挥重要作用的吡啶环β位单加氧酶：恶臭假单胞菌S16菌株的吡啶环β位单加氧酶HspB、HspA以及恶臭假单胞菌KT2440菌株的吡啶环β位单加氧酶NicC。我们经过艰苦努力，解析了恶臭假单胞菌S16菌株的吡啶环β位单加氧酶HspB的晶体结构，分辨率为2.1埃。通过计算机辅助虚拟对接的方法，得到了HspB与其辅基FAD和其底物HSP的复合物的结构模型，并进一步揭示了HspB结合辅基FAD和底物HSP的分子机理，通过突变体的酶学检测进行了证实。我们还通过对HspB的分子动力学模拟研究，发现HspB的β2-α2 loop和β18-α9 loop存在着较大的柔性，可能与HspB在催化反应过程中将底物和辅基进行位置调整从而进行精准对接的柔性有关。我们的这项工作正在投稿国际微生物学领域顶级期刊Molecular Microbiology。另外，我们还解析了恶臭假单胞菌KT2440菌株的吡啶环β位单加氧酶NicC与其辅基FAD的复合物的晶体结构，分辨率为1.75埃，正在继续进一步进行NicC与底物的共结晶研究，并进行深度机理研究。此外，我们还对恶臭假单胞菌S16菌株的吡啶环β位单加氧酶HspA进行了纯化与结晶研究。在本项目的支持下，发表SCI论文10篇，其中本人作为通讯作者的论文8篇，申请国际专利一项。2017年2月，荣获国家教育部高等学校科学研究优秀成果奖（科学技术）自然科学奖一等奖，本人排名第四。2019年，本人代表上海交通大学承办了第六届华东结构生物学会议。2020年，本人当选上海生物物理学会理事。在本项目资助下，培养3名博士生毕业，培养4名硕士生毕业。

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