芳香杂环环境污染物微生物降解途径中的关键酶—钼喋呤依赖型多亚基单加氧酶Spm的结构与功能研究

Heterocyclic compounds are the major environmental pollutants in our country, and cause many human diseases such as tumors and fetal malformations. The microbiological technique is one of the most efficient approaches to control environmental pollution caused by heterocyclic compounds. In microbial catabolic pathways of aromatic compounds, using a monooxygenase to catalyze the first hydroxylation modification on the aromatic rings is a key step which activates the chemically inert aromatic ring. For N-heterocyclic compounds,this hydroxylation reaction on the N-heterocyclic rings is usually catalyzed by a multi-subunit molybdopterin-dependent monooxygenase. However, until now we only have limited information on the structures and catalytic mechanisms of these important monooxygenases. In this proposal, we plan to study the 3-succinyl pyridine monooxygenase Spm in the nicotine degradation pathway of Pseudomonas putida S16, which catalyzes the first hydroxylation reaction on the pyridine ring of nicotine in this pathway. The Spm holoenzyme consists of three subunits: SpmA which carries a molybdopterin cofactor, SpmB which carries an FAD cofactor, and SpmC which carries a [2Fe-2S] iron-sulfur cluster. We plan to determine the crystal structure of the Spm holoenzyme as well as the structure of Spm in complex with its substrate 3-succinyl pyridine. Using the crystal structure of the enzyme-substrate complex as the initial coordinates, we are going to carry out computer molecular dynamics simulation and quantum chemical calculation to elucidate the molecular mechanism of how Spm catalyzes the hydroxylation modification on the heterocyclic ring, which is a difficult chemical reaction in nature. Through site-directed mutagenesis and measurement of enzymatic activities, we are going to investigate the roles of key active site amino acid residues of Spm in its subunit assembly, in its cofactor binding, in its substrate recognition, as well as in the reaction catalysis process. By expression of mutant plasmids in the spm gene knockout bacteria strain, we are also going to study how mutating these vital amino acid residues of Spm would affect the metabolic functions of Pseudomonas putida S16. Through the study of Spm, we are going to investigate important properties of multi-component molybdopterin-dependent monooxygenases in microbial degradation pathways of heterocyclic pollutants.

芳香杂环化合物是重要的环境污染物和环境毒物。在微生物降解芳香类化合物的代谢途径中，以单加氧酶催化芳香环上的第一步羟基化以活化芳香环是一个关键步骤。对氮杂环化合物而言，一般由钼喋呤依赖型多亚基单加氧酶催化氮杂环上第一步羟基化，但人们对这类酶的结构与机理的认识不足。本项目中，我们以恶臭假单胞菌中催化氮杂环─吡啶环上第一步羟基化的三亚基含钼喋呤、铁硫簇、FAD辅基的3-琥珀酰吡啶单加氧酶Spm为研究对象，计划：1）解析其全酶复合物及与底物的复合物的晶体结构，揭示其三个亚基与三个辅基装配成全酶及特异性识别底物的分子机制；2）在结构基础上进行分子动力学模拟与量子化学计算，阐明其催化芳香杂环上羟基化的分子机理；3）通过突变体酶活性检测与体内基因敲除/回补实验，考察结构所揭示的重要残基对酶活性与代谢功能的关键作用。通过对Spm的研究，我们将探讨微生物代谢杂环污染物的钼喋呤依赖型多亚基单加氧酶的重要性质。

钼喋呤依赖性三亚基单加氧酶在微生物降解含氮环境毒物的代谢途径中起着重要作用。我们解析了与Spm非常类似的、节杆菌的尼古丁降解代谢途径中关键的钼喋呤依赖性三亚基单加氧酶Kdh全酶复合物的晶体结构。与Spm一样，Kdh的大亚基KdhL、中亚基KdhM、小亚基KdhS也分别含有钼喋呤胞嘧啶二核苷酸、黄素腺嘌呤二核苷酸FAD、[2Fe-2S]铁硫簇辅基。Kdh催化与Spm类似的尼古丁的吡啶环上的单加氧反应，将6-羟基假氧化尼古丁氧化生成2,6-二羟基假氧化尼古丁。我们将节杆菌Kdh的三个亚基的编码基因克隆到节杆菌与大肠杆菌的穿梭质粒pART2中，在节杆菌中共表达，纯化得到了Kdh全酶复合物并将其结晶。通过在上海光源收集的数据，解析了Kdh三亚基复合物（包括大亚基KdhL及其所结合的辅基钼喋呤胞嘧啶二核苷酸、中亚基KdhM及其所结合的辅基黄素腺嘌呤二核苷酸FAD、小亚基KdhS及其所结合的辅基[2Fe-2S]铁硫簇）的晶体结构，分辨率为3.4埃。我们发现在KdhL和KdhM分别位于两侧，把KdhS夹在中间。因此KdhL与KdhS一起把钼喋呤胞嘧啶二核苷酸紧密包裹在Kdh全酶内部，可能防止氧气进入活性中心产生副反应。与之相反，KdhM的辅基FAD处于一个相对开放的位置，不是被紧紧包裹的，这可能与FAD在还原为FADH2后，需要被立即氧化，再次进入反应循环有关。在KdhS中，结合着两个[2Fe-2S]铁硫簇。KdhS被KdhL和KdhM夹在中间，从而在它们的协助下，把两个[2Fe-2S]铁硫簇包裹在其内部，防止被氧气氧化。在Kdh全酶复合物中，KdhL、KdhS、KdhM携带的三种辅基成线性排列，有利于电子的传递。通过计算机模拟对接，我们得到了Kdh与底物6-羟基假氧化尼古丁的复合物的预测结构。在该结构中，底物与大亚基KdhL所结合的辅基钼喋呤胞嘧啶二核苷酸的距离为4.5埃。各个辅基（钼喋呤胞嘧啶二核苷酸、两个铁硫簇、FAD）之间的距离都在10-14埃左右，距离适中，在量子化学理论上电子所能穿透的能垒宽度之内。由于该体系较复杂，我们还在对该体系进行量子化学计算，探讨Kdh的催化机理，并对Kdh催化底物反应的酶反应动力学常数KM、kcat进行测量。发表SCI论文14篇（通讯作者论文9篇）。申请中国专利和国际专利各一项。2016年获教育部高等学校科学研究优秀成果自然科学奖一等奖。