新型吲哚异戊烯基转移酶DmtC1的底物宽泛性及催化分子机制

The aromatic prenyltransferases transfer prenyl moieties from isoprenoid donors to various aromatic acceptors, which can greatly enrich the structural diversity of compounds and significantly enhance their biological activities. Therefore, the novel aromatic prenyltransferases receive extensive attention, especially those with substrate promiscuity. In previous study, a unique phytoene-synthase-like (PSL) prenyltransferase DmtC1 was characterized in a tRNA-dependent diketopiperazine-terpene biosynthetic pathway, which shows no homology to typical soluble aromatic prenyltransferases. Based on the novelty of DmtC1, this project aims to carry out the following investigations: exhaustive analysis of the biochemical properties and substrate promiscuity of DmtC1; using site-directed mutagenesis and combining with protein structure analysis to reveal the catalytic machinery of DmtC1. Our investigation will not only provide new tool enzyme for chemoenzymatic synthesis of prenylated aromatic compounds which will set the theoretical foundation for enzyme engineering, but also provide numerous lead compounds for our country’s new drug discovery and development.

芳香族异戊烯基转移酶负责催化芳香前体化合物中引入异戊烯基，能极大丰富化合物结构多样性，显著提高生物活性，因而新颖芳香族异戊烯基转移酶的发掘及其底物宽泛性研究备受关注。前期，本研究组揭示了一类tRNA依赖型的二酮哌嗪-萜类化合物生物合成机制，其中萜烯化是由一个独特的、与已鉴定芳香族异戊烯转移酶无同源性的PSL (phytoene-synthase-like) 型异戊烯基转移酶DmtC1负责组装而成。基于该酶的新颖性，本项目拟进一步采用体外生化反应等手段，对DmtC1进行酶学表征，挖掘其底物宽泛性；采用定点突变及晶体学手段，揭示其分子催化机制。一方面有望将其开发新型工具酶用于芳香族杂萜类化合物的结构多样性研究，为开展酶工程研究提供理论基础；另一方面将提供更多的先导化合物实体，对促进具有我国自主知识产权的新药研发，具有重要的理论意义和应用价值。

新型芳香族异戊烯基转移酶对异戊烯化天然产物结构多样性研究具有重要意义。PSL (phytoene-synthase-like)型异戊烯基转移酶DmtC1 能以多种含色氨酸环二肽为底物催化C-3 “regular”异戊烯化反应，具有底物宽泛性。本项目以DmtC1为研究对象，1. 对DmtC1的底物宽泛性及酶学性质进行深入研究，DmtC1能够识别17种环二肽和3种C5-C20的异戊烯基供体，揭示了 DmtC1 为首个具有底物宽泛性的PSL型家族新成员；2.利用DmtC1催化不同环二肽发生异戊烯基化反应，酶法合成得到42个pre-drimentines类化合物；3. 预测了DmtC1的三维结构，通过定点突变研究探索了DmtC1的催化机制；另外开展了DmtC1同源蛋白的功能及所处基因簇的激活、活性化合物及其生物合成的研究工作。本项目一方面加深了我们对PSL型异戊烯基转移酶家族的认识，为采用化学酶法和组合生物合成策略进行异戊烯化二酮哌嗪类化合物结构多样性研究提供了科学理论指导；另一方面为新药研发提供更多的先到化合物实体，促进形成具有我国自主知识产权的新药具有重要的理论意义和应用价值。