新颖具杂泛性di-C-糖基转移酶MiCGTb催化机制及应用研究

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.

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。

C-糖苷是活性天然产物及临床药物中一类非常重要的化合物。但天然C-糖苷类化合物发现较少，而di-C-糖苷类化合物的种类及数量则更少。近年来化学法C-糖基化取得重要进展，但区域及立体选择性地合成di-C-糖苷仍具挑战性。酶法C-糖基化具有高度选择性，而且环境友好，为di-C-糖苷类化合物的高效合成提供新策略。在前期研究中，我们发现C-糖基转移酶MiCGTb能催化di-C-糖基化反应，但其活性位点及催化机制尚未阐明。本项目利用蛋白结构域交换及点突变策略，发现MiCGTb中Ile152是负责di-C-糖基化反应的活性位点，His23是C-糖基化反应的催化位点，Ser60/Val100/Thr104与di-C-糖基化反应活性相关。分子动力学表明，Ile152位于糖基受体结合腔底部，不同氨基酸取代可能会引起结合腔形状或大小改变，进而控制着二次C-糖基化反应发生，首次揭示了di-C-糖基化反应的酶学机制。对MiCGTb中Ser60/Val100/Thr104/Ile152四个活性位点进行理性设计，构建了具有催化活性高、底物谱(尤其是糖基供体)宽的突变体酶库。突变体MiCGTb-GAGM能够催化11个不同的α-D-和β-L-糖基供体形成结构新颖、含不同构型C-糖基的糖苷类化合物，这是目前报道的催化糖基供体最多的C-糖基转移酶。而且利用MiCGTb-GAGM首次实现稀有活性香豆素C-糖苷酶法合成，为香豆素类C-糖苷的合成提供了新策略。通过药理活性评价，发现多个C-糖苷具有细胞毒活性和SGLT2抑制活性，为创新药物研发提供先导化合物，具重要理论意义及实际应用价值。. 本项目在实施过程中，以第一作者在ACS Catalysis、Organic Letters、Tetrahedron Letters等杂志发表学术论文4篇（均标注基金资助），其中3篇为SCI收录论文，单篇最高影响因子12.35。