Pseudomonas putida DLL-E4对硝基苯酚-4-单加氧酶PnpA催化机制的研究

p-nitrophenol (PNP) is an important aromatic pollutant. It has been on the list of "preferred controlled pollutant" in China and the United States. Pseudomonas putida DLL-E4 can efficiently degrade PNP. It contains all genes involved in the PNP degradation. Gene pnpA encodes the p-nitrophenol-4-monooxygenase, PnpA, which catalyzes the first step of PNP degradation. Our previous study showed that there are many differences in the protein structure and enzyme properties between PnpA and other PNP monooxygenase. Thus, we speculated that the catalytic process of PnpA is different from other conventional PNP monooxygenase. So far, we have obtained the crystals of apo-PnpA, PnpA-PNP, PnpA-FAD-PNP, and Se-PnpA, and analyzed their structures. The preliminary three-dimensional structure model of PnpA has been built. However, the detailed information about structure and its key sites are still obscure. In this project, we plan to refine the structural model of PnpA by using CCP4, Coot, Phenix and other softwares. The structural model of PnpA-PNP and PnpA-NADH complex are going to be proposed through Autodock. We plan to confirm the substrate PNP binding sites, the cofactor FAD binding sites, the cofactor NADH binding sites, and the catalytic sites by site-direct mutation according to the determined structural information. Based on the result of site-direct mutation and structure determination, the molecular mechanism of PnpA catalyzing PNP to p-benzoquinone and nitrite could be clarified.

对硝基苯酚（PNP）被列入“优先控制污染物名单”，可被Pseudomonas putida DLL-E4高效降解。菌株DLL-E4含有对硝基苯酚-4-单加氧酶PnpA，该酶可催化PNP脱硝基反应。前期我们发现PnpA与其他PNP单加氧酶在结构、酶学性质上存在差异，因此推测其催化过程不同于其他常规PNP单加氧酶。前期我们已得到多种形式的PnpA蛋白晶体及晶体结构初步模型。本项目拟采用CCP4、Coot、Phenix等软件对初步结构模型进行修正，并利用分子对接法获取PnpA-底物复合物结构模型。根据所得到的结构信息对底物和辅因子结合位点、催化中心等关键位点进行定点突变验证，并分析野生蛋白与突变蛋白之间的酶动力学差异以及突变蛋白晶体的结构变化。本项目的研究意义在于从晶体结构角度分析PnpA与其他PNP单加氧酶在结构、酶学性质上的差异原因，从而提出PnpA催化PNP生成对苯二醌和亚硝酸根的分子机制

Pseudomonas putida DLL-E4可高效降解对硝基苯酚（PNP），其体内含有对硝基苯酚-4-单加氧酶PnpA，该酶可催化PNP脱硝基反应。本项目采用晶体结构解析、蛋白定点突变、基因敲除、蛋白互作等手段研究PnpA的晶体结构、PnpA的关键位点、Crc对PnpA的作用，以此来解析PnpA催化PNP形成对苯二醌的催化机制。. 项目组从以上三方面开展研究，并取得相关研究成果：（1）PNP位于蛋白质内部异咯嗪环的N5边缘附近，PNP的硝基直接与Val54和Arg234形成氢键，而羟基与Val223和Cys236形成氢键；（2）Glu42、Gln115、Arg179、Arg282、Asp297或Pro304的突变皆可引起酶催化效率的下降；（3）预测了PnpA的催化机制：在蛋白质内部，辅因子FAD在NADH和氧分子作用下形成异咯嗪C4a -过氧化氢中间体，随后异咯嗪C4a -过氧化氢中间体的羟基通过亲电攻击与PNP的C4发生反应，使得亚硝酸根被释放。亚硝酸根释放后形成的中间体在异咯嗪C4a -过氧化氢中间体的氧原子作用下发生去质子化而形成对苯二醌，同时FAD恢复原来的结构；（4）crc的敲除使菌株降解PNP的速度明显加快，Crc与PnpA之间存在互作，表明Crc对PnpA具有一定的调控作用。. 本项目的研究成果为我们理解微生物来源的降解酶降解污染物提供理论基础，为改造微生物来源降解酶和修复环境污染提供技术指导。