红花醌式查尔酮碳苷糖基转移酶的功能鉴定及其生物催化应用

C-glycosylation is a relatively rare type of glycosylations in natural products. It not only enriches the structural diversity of natural products but also improves the water solubility, pharmacological activity and druggability. However, C-glycosylated natural products are fairly limited and difficult to synthesize by chemical methods. Safflor yellow, the main bioactive constituent of Carthamus tinctorius, is composed of a series of C-glycosylated quinochalcones. However, the key C-glycosyltransferase (CGT), as well as the catalytic mechanism, involed in the biosynthetic pathway of safflor yellow remains unknown. In our previous work, 34 candidate genes of CGTs have been successfully cloned from C. tinctorius via transcriptome sequencing, homology of CGTs and rapid-amplification of cDNA ends (RACE). Based on these, this proposal is to screen and identify the CGT from the candidate genes and thus to uncover the C-glycosylation in quinochalcone C-glycosides biosynthesis. Guided by homology modeling 3D structures of the CGT and site-directed mutagenesis, the catalytic active motif, the mechanism of C-glycosylation, and substrate structure-enzyme function relationships will be clarified. Furthermore, a library of mutants of the CGT with substrate promiscuity will be constructed to establish a C-glycosylation platform of C-glycosyldiversification of bioactive natural products for the discovery of drug leads or candidates. Therefore, this study of CGTs from C. tinctorius is of importance both in theory and practice.

C-糖基化是天然界中较为罕见的一种糖基化形式，不仅能丰富天然产物的结构多样性，而且能改善其水溶性及药理活性等，提高成药性。但是，天然C-糖苷类化合物十分有限，化学合成困难。中药红花的活性成分红花黄色素为结构独特的醌式查尔酮C-苷类化合物，催化其形成的关键酶C-糖基转移酶尚未被发现，C-糖基化机制亦未明晰。前期通过转录组测序，利用植物C-糖基转移酶的同源性与RACE技术，获得了34个C-糖基转移酶候选基因。基此，本研究拟从候选基因中寻找C-糖基转移酶，揭示红花黄色素生物合成的C-糖基化机制；在蛋白三维结构同源模建指导下深入研究红花C-糖基转移酶活性结构域，针对活性位点进行定点突变等，探讨催化机制、功能与底物结构之间的关系以及人工设计构建突变体酶，拓宽底物谱，建立活性天然产物C-糖基化新策略，催化合成结构新颖多样的活性C-糖苷类化合物，为药物先导物/候选物发现奠定基础，具理论及实际应用意义。

糖基化反应可以增加化合物水溶性，降低化合物的毒副作用，提高很多化合物的生物活性。酶法糖基化具有反应条件温和、较高的转化率和区域选择性等优点，因此酶法糖基化成为目前糖基化修饰的研究热点。C-糖基化是天然界中较为罕见的一种糖基化形式，并且天然C-糖苷类化合物十分有限，化学合成困难。中药红花的活性成分红花黄色素为结构独特的醌式查尔酮C-苷类化合物，催化其形成的关键酶C-糖基转移酶尚未被发现，C-糖基化机制亦未明晰。我们从红花的新鲜花冠中克隆获得1个全新的C-糖基转移酶基因CtGTL1，并成功进行了外源表达及重组蛋白纯化。体外酶促反应检测发现CtGTL1不仅具有单C-糖基化活性还表现出双C-糖基化活性，即在苷元分子上引入两分子C-葡萄糖基，并具有较高的催化效率。通过对该C-糖基转移酶的底物谱进行研究，发现其具有较为宽泛的底物谱，为后续开发为C-糖基化工具酶奠定了基础。另外，在糖基转移酶活性筛选中我们还发现了3个新颖O-糖基转移酶能够高效催化小檗碱类化合物进行糖基化修饰；糖基转移酶UGT71E5的N-糖基化活性具有较强的底物杂泛性，并首次发现该酶能够催化多种N-杂环芳香类化合物形成N-糖苷。上述研究为药物先导物/候选物发现奠定基础，具理论及实际应用意义。.本课题在实施过程中，已发表学术论文3篇（均标注基金资助），均为SCI收录论文，包括Advanced Synthesis & Catalysis、Chinese Chemical Letters、Tetrahedron Letters本领域期刊。