库拉索芦荟中芦荟苷生物合成关键酶C-糖基转移酶的功能鉴定及其C-糖基化应用

C-glycosyltransferases (CGTs) are responsiable for the transferring sugars from donors to acceptors and generating rare C-C glycosidic bonds, which possess great application prospects in directional C-glycosylaton. There are only a few of CGTs have been identified and the catalytic mechanism remains unclear. Aloin, the main bioactive constituent of Aloe barbadensis Miller, is C-glycosylated anthrone. However, the key CGTs which are responsiable for the biosynthesis of C-glycosylated anthrones have not been identified. In our previous work, 52 candidate genes of CGTs have been successfully cloned from A. barbadensis via transcriptome sequencing, homology of reported CGTs and rapid-amplification of cDNA ends (RACE). On this basis, this study plans to screen and identify the anthrones CGT from the candidate genes and thus to uncover the key step of Aloin biosynthesis that is the mechanism of C-glucose introduction. Under the guidance of homology modeling and site-directed mutagenesis, the catalytic active motif of the CGT will be studyed to explore the mechanism of C-glycosylation and substrate structure-enzyme function relationships. Furthermore, a library of mutants of the CGT will be prepared with rational design to establish novel and designable methods of C-glycosylation with independent intellectual property rights. It not only possesses important theory significance but also has practical application significance in utilizing the enzymatic methods to efficiently synthesis bioactive natural/unnatural C-glycosides providing lead molecules for drug discovery.

C-糖基转移酶负责将糖基由供体转移到受体分子上形成稀有的C-C糖苷键，在活性天然产物的定向C-糖基化修饰领域中具有巨大的应用潜力。目前仅有少数C-糖基转移酶被发现，且催化机制尚未完全阐明。中药库拉索芦荟的活性成分芦荟苷为结构独特的蒽酮C-苷类化合物，负责其合成的关键C-糖基转移酶尚未被发现。前期工作中，通过转录组测序、蛋白同源性及RACE技术，获得了52个C-糖基转移酶候选基因。在此基础上，本研究拟从候选基因中对C-糖基转移酶进行功能鉴定，揭示芦荟苷生物合成途径中的关键步骤C-糖基化的机制。利用同源建模结合位点突变，研究C-糖基转移酶活性结构域，阐明蒽醌C-糖基转移酶的催化机制、功能与底物结构之间的关系，进而理性构建突变体酶，建立具自主知识产权的定向C-糖基化新方法。利用酶法高效合成结构新颖多样的活性天然/非天然C-糖苷类化合物，为药物先导物发现奠定基础，具理论意义及实际应用价值。

C-糖基转移酶负责将糖基由供体转移到受体分子上形成稀有的C-C糖苷键，在活性天然产物的定向C-糖基化修饰领域中具有巨大的应用潜力。目前仅有少数C-糖基转移酶被发现，且催化机制尚未完全阐明。在前期工作基础上，本研究从库拉索芦荟中鉴定了一个新颖的C-糖基转移酶（AbGT73），该酶能够催化多种不同类型的药物类似物受体及糖基供体分别生成C-, O-, N-与S-糖苷。更重要的是，AbGT73能够高效催化已知糖基转移酶无法催化的受体苷元进行C-糖基化。基于AbGT73的催化杂泛性与高效性，我们利用化学-酶法高效合成了16个新颖且具有SGLT2抑制活性的C-糖苷。值得注意的是，其中C-糖苷46a显现出良好的体内抗糖尿病活性。同时，建立了全细胞催化法，C-糖苷46a的产量可达克级/升，并且无需添加昂贵的糖基供体。此外，通过对AbGT73的同源建模、分子对接以及定点突变，获得了1个与AbGT73催化C-/O-选择性相关的关键氨基酸位点。通过对该位点的饱和突变，提高了AbGT73催化C-糖基化的选择性。这些发现不仅表明AbGT73可开发作为药物开发与合成中的工具酶，还暗示AbGT73是研究C-糖基转移酶催化机制与改造C-糖基转移酶选择性的理想材料。