海洋细菌Rhodococcus rhodochrous新型水合酶的挖掘及其催化性质与机制

Chiral alcohols are key intermediates in the synthesis of various important pharmaceutical products, indeed, 40% of which contain chiral alcohol as the essential structures. Biocatalytic asymmetric hydration of alkenes represents a highly attractive reaction to chiral alcohols, however, most of the known hydratase enzymes are part of the metabolic pathways and therefore exhibit a very narrow substrate scope. Marine ecological environments make the marine enzymes different from terrestrial ones in substrate specificity and stereoselectivity, which show high potentials as biocatalysts. In previous work we reported a strain of bacteria isolated from Pacific Ocean seawater catalyzing the hydration of alkenes to produce chiral alcohols with high enantioselectivities and high yields. We have proved that the newly discovered bacteria catalyzed the hydration of alkenes with a very broad and new substrates scope, implying the novelty of the marine hydratase. In this project, we aim to isolate the novel hydratases from the newly discovered marine-derived bacteria and investigate the unique mechanisms responsible for the broad substrates scope. We have found three putative hydratases expectedly by genome hunting expectedly. In this project, we first will identify the gene functions of the three putative hydratases by gene knockout and complement technologies. Based on the results, all of the three (or one or two) hydratases will be cloned, purified and fully characterized for their chemical and biological properties. Specially, a model of the hydratases, built by homology modeling, will be docked with various substrates to identify the interaction between them, demonstrating the catalytic mechanism. The results will provide a novel biocatalyst for synthesis of chiral alcohol and the theoretical basis for rational design of hydratases.

手性醇是医药合成时的关键中间体，约40%的药物活性分子含有手性醇结构。酶催化烯烃不对称水合反应是制备手性醇的最佳途径之一，但目前已知的水合酶大部分只催化特定的天然底物，严重影响了其应用。海洋微生物由于其体内特殊的代谢途径和酶系，是挖掘新颖酶催化剂的重要来源。申请者前期工作报道了一株分离自太平洋海水的细菌高效高选择性催化烯烃不对称水合反应，其催化的底物结构是新的、底物谱是宽的。这表明该菌体内负责催化作用的水合酶是新的，其特有的宽底物谱的机制是新的。申请者发现该菌基因组存在三个水合酶基因。本项目拟进一步采用基因敲除与回补、基因异源表达和实时定量PCR等方法分别研究水合酶基因产物的活性、酶学性质、基因表达调控和生理功能，尤其对底物谱的影响。本项目旨在获取具有宽底物谱的海洋水合酶并研究其催化性质及机制，为手性醇的生物合成提供更高效的催化剂，为水合酶的分子理性改造和功能强化提供理论依据。

本项目首先对项目前期工作获取的三个水合酶候选基因（命名为Rhr-HY1、Rhr-HY2、Rhr-HY3）进行信息分析，发现它们具有催化烯烃水合反应特定的活性部位，且与已知的具有该催化活性的酶基因具有一定的同源性。但是Rhodococcus rhodochrous的催化活性是单个候选酶的作用结果还是三个候选酶共同作用的结果仍并不清楚；另一方面Rhodococcus rhodochrous特有的宽底物谱是三个候选酶累加起来的结果还是它们分别能催化所有底物也并不清楚，更多的细节需要实验来验证。实验采用基因敲除和基因回补技术分别鉴定了这三个候选水合酶基因的体内功能。实验结果显示候选水合酶Rhr-HY3的基因敲除突变株和基因回补后的突变株对底物3-甲基-2（5H）-呋喃酮表现出明显差异；而候选基因Rhr-HY1和Rhr-HY2的基因敲除突变株和基因回补突变株对相同底物并没有明显差异，据此推断候选基因Rhr-HY3可能为负责催化水合反应的新型酶。接着，对水合酶Rhr-HY3进行异源表达和分离纯化，获得了大量重组的水合酶，并以化合物3-甲基-2（5H）-呋喃酮为标准底物，验证了重组酶的体外活性，进一步证实海洋细菌Rhodococcus rhodochrous催化水对烯烃不对称加成反应主要依赖水合酶Rhr-HY3得以实现。最后，对水合酶Rhr-HY3的酶学性质（包括温度、pH、金属离子、有机溶剂对酶活的影响）、底物特异性及立体选择性（包括底物分子的空间构型及电荷分布与酶活性及立体选择性的相互关系）、动力学参数（Vmax、Km、Kcat）进行详细表征。本项目基本按照研究计划完成，研究成果已经申请并获得授权中国发明专利2项（项目负责人均为第一发明人）；4篇SCI学术论文（项目负责人为通讯作者，本项目为第一标注的SCI论文1篇；项目负责人为第一作者，本项目为第一标注的SCI论文1篇；项目负责人为通讯作者，本项目为第二标注的SCI论文1篇；项目负责人为通讯作者，本项目为第三标注的SCI论文1篇）；并获得1项省级后续资助项目（广东省自然科学基金面上项目，项目负责人为主持人）。