碳磷裂解酶参与的碳磷键断裂自由基反应机制研究

Along with more and more phosphonates used in human activities, water and soil pollution become a serious issue due to the stable C-P bond structure in the compounds. There is an urgent need to develop biodegradable phophonates pathway. There are 4 reported enzymes that are capable to degrade phosphonates. C-P lyase catalyze a broad of phonsphonates and is the ideal candidate for biodegrading C-P bond compounds. Recent studies have shown that PhnJ, the key enzyme in the C-P lyase complex, can only complete 1-4 catalytic turnovers. It's activity is low and unstable, which greatly limits the catalytic efficiency of the whole pathway. In this proposal, the core catalytic center, iron-sulfur cluster will be studied with the focus of redox chemistry. The detail radical reaction mechanism and the key intermediate, protein based thio-phosphonate, in the PhnJ reaction will be investigated. The proten-protein interaction between PhnJ and other memebers of the C-P lyase will be evaluated as well. Optimized activity and completed radical mechanism will be proposed based on above studies. This project achievements will not only elute the more clear reaction mechanism and the structure of active site, but also pave the path for solving the man-made phosphonate pollutions with enzyme-engineering or synthetic biology tools. In addition, the studies will also provide the new mentality and the new strategy for the bioorganic chemistry research, promote the radical SAM iron-sulf cluster metallo-proteins research level in China.

有机膦化合物在造福人类活动的同时，其稳定碳磷键结构的难降解性所造成的水和土壤污染问题日益严重，迫切需要发展有机膦生物降解的方法。在迄今报道的四种可降解有机膦化合物的生物酶中，碳磷裂解酶复合体对底物的适用性最宽泛，可有效催化各种有机膦化合物中的碳磷键断裂，是理想的有机膦生物降解酶。最新的研究表明此酶复合体中的关键酶PhnJ仅可以完成1-4个催化循环，活性低且不稳定，大大限制了整个途径的催化效率。本申请以核心的催化中心铁硫簇体为切入点，分析其还原再生效率的影响因素，解析反应过程的自由基催化机制和关键硫代膦酸盐反应中间体，考察碳磷裂解酶复合体中其他酶与PhnJ的相互作用和活性影响。绘制完善的自由基反应催化机理并获得优化的酶催化活性，丰富这一重要自由基S-腺苷甲硫氨酸蛋白家族的新颖生物化学反应，为利用生物工程和合成生物学来解决有机膦环境污染问题提供应用研究基础。

本课题以核苷类抗生素多氧霉素为研究对象, 通过体内敲除和体外重构 polH和polJ蛋白的方式, 利用PolH-PolJ体外双酶偶联反应体系和PolH单独反应体系确认PolH以3'-EUMP 为底物并生成独特八碳糖双环结构庚糖酸-5'-磷酸 octosyl acid 5'-phosphate (OAP)的关环产物, OAP在磷酸酶PolJ的作用下脱磷酸生成庚糖酸octosyl acid(OA), 从而解析了核苷类抗生素重要骨架OA的合成途径。.通过PolH在氘水中的反应及所有半胱氨基酸突变体活力测试, 发现第 C209位的蛋白能够为底物自由基中间体提供还原的氢原子, 生成OAP及其异构体epi-OAP。利用同位素底物[5'5"-2H2]-3'-EUMP, 发现SAM还原形成的5'-dA自由基首先攫取底物C5'位的氢形成底物自由基中间体, 然后自由基电子转移到C7'位并最终被209位的半胱氨酸还原形成OAP和epi-OAP。PolH很有可能仅有一个铁硫簇参与电子传递, 进而初步解析了 SAM自由基酶PolH的催化机制。.综上，本研究鉴定了多氧霉素合成途径中的PolH和PolJ两个关键酶的功能,解析了核苷类抗生素重要骨架OA的合成途径, 大大地推动了多氧霉素及其他类似骨架核苷类抗生素生物合成途径解析的研究进展; 深入研究了SAM自由基酶 PolH的催化机制, 为首个清晰解析的通过氨基酸还原自由基并引发C-C键形成的蛋白, 对于丰富SAM自由基酶的催化机制类型有重要的意义。