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。

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.