天然di-C-糖苷类化合物种类及数量稀少，化学合成困难；而已发现的C-糖基转移酶仅有mono-C-糖基化活性，且糖基供体宽泛性不足，导致di-C-糖苷类化合物来源受限，结构多样性不足。本课题组前期从芒果中克隆得到两新颖C-糖基转移酶MiCGT和MiCGTb，功能研究表明MiCGTb具有di-C-糖基化活性，而MiCGT仅能催化形成mono-C-糖苷，且MiCGTb具有很强的糖基供体杂泛性，但其活性位点及催化机制尚未阐明。本项目拟在此基础上，利用mono-C-糖基转移酶与di-C-糖基转移酶空间结构差异性，结合结构域交换及点突变策略，解析di-C-糖基转移酶的活性位点及催化机理；进一步利用活性位点饱和突变及多位点联合突变对酶进行理性设计，获得能催化不同苷元受体与单糖供体的突变体酶，建立具自主知识产权、高效、可设计性di-C-糖基化新策略，为药物发现提供先导分子，具重要理论创新及实际应用价值。

The structural diversity and abundance of naturally occurring di-C-glycosides appear to be sparse, while it is also difficult to access via chemical synthesis; moreover, the known C-glycosyltransferases (CGTs) exhibited only mono-C-glycosylation activity and relatively narrow sugar donor spectra, thus limiting the availability of diverse di-C-glycosides. In our previous investigation, two novel CGTs MiCGT and MiCGTb were cloned from Mangifera indica. Functional studies indicated that MiCGTb has the capacity of di-C-glycosylation, whereas MiCGT only catalyzes the formation of mono-C-glycosides; and MiCGTb shows a glycosyl donor promiscuity, however，its active sites and catalytic mechanism of di-C-glycosylation remain unclear. Based on these interesting and promising findings, this proposal is to focus on investigation on the active regions, sites and catalytic mechanism of di-CGT by analyzing the structural differences between mono-CGT and di-CGT, combined with domain exchange and site mutation strategies; rational design of di-CGT will be further performed to broaden the substrate-spectrum (donor/acceptor) of enzyme mutants by using the saturation mutation and multi-site co-mutation strategy. These works will establish a powerful and designable di-C-glycosylation platform for the discovery of drug leads with independent intellectual property. This project is established in self-dependent innovation, and its outcome is of importance in both theory and practice.