源自芒果一新颖具杂泛性C-糖基转移酶MiUGT13的结构与功能研究

C-glycosylation can not only increase the bioavailability and pharmacological activity of compounds, but also enhance their stability and prolong their period of validity due to their more stable glycosidic bond and molecules. Therefore, C-glycosylation can improve the druggability of the compounds, and is an important modification reaction in drug R & D. However, the availability of C-glycosides is obviously limited because of the rare occurrence in nature and difficult access via chemical synthesis. Furthermore, the C-glycosylation by employing C-glycosyltransferase (CGT) is restricted by the limited CGTs, unclear catalytic mechanism, and relatively narrow substrate spectra. In our previous investigation, a new gene, MiUGT13 from Mangifera indica, was cloned. Functional characterization revealed that the recombinant MiUGT13 can efficiently catalyze the C-glycosylation of maclurin, a putative precursor of mangiferin, which illustrating the biosynthesis pathway of mangiferin. Interestingly, it was found that MiUGT13 displayed robust substrate promiscuity. Based on these interesting and promising findings, this proposal is to focus on further investigation on the substrate spectra of MiUGT13, while revealing its active site, catalytic mechanism, and structural basis of promiscuity through structural biology combined with site-directed mutation. Moreover, structure-guided manipulation of MiUGT13 will be performed for the purpose of broadening the substrate-spectrum (donor/acceptor) of enzyme mutants. These works will establish a powerful, designable, and chemo-enzymatic C-glycosylation platform of bioactive natural/unnatural compounds 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.

C-糖苷化不仅能增加化合物的生物利用度和药理活性，且糖苷键不易断裂、结构稳定，使其具稳定和持久的药效，提升其成药性，在药物研发中具有重要意义。天然C-苷类化合物数量少，结构多样性不足，化学合成因选择性等不足而不易获得。迄今已发现的C-糖基转移酶较少，催化机制不明且底物谱窄，应用受限。前期研究中，我们从芒果中克隆得到一全新C-糖基转移酶基因MiUGT13，功能验证表明重组MiUGT13能高效催化芒果苷的前体桑橙素C-糖基化，阐明了芒果苷的生源途径，并发现该酶具很强的底物杂泛性。在此基础上，本项研究拟进一步考察该酶底物谱，通过结构生物学与定点突变策略解析其活性区域，揭示其催化机理及杂泛性结构基础；并在结构导向下设计构建突变体酶，拓宽其底物（受体/供体）谱，建立具自主知识产权、高效、可设计性结构多样天然/非天然活性化合物的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-糖苷化不仅能增加化合物的生物利用度和药理活性，且糖苷键不易断裂、结构稳定，使其具稳定和持久的药效，提升其成药性，在药物研发中具有重要意义。天然C-苷类化合物数量少，结构多样性不足，化学合成因选择性等不足而不易获得。迄今已发现的C-糖基转移酶较少，催化机制不明且底物谱窄，应用受限。.在本研究中，我们从芒果叶中克隆获得了2个全新的C-糖基转移酶基因（MiUGT13或MiCGT，MiCGTb）并进行了异源表达，功能鉴定表明它们均能催化芒果苷前体桑橙素二苯甲酮化合物形成C-苷，为其生物合成相关关键酶。进一步底物谱研究表明，MiCGT及MiCGTb均具有宽泛的受体底物谱。有趣的是，前者仅具单C-糖基化活性，而后者还具bis-C-糖基化活性。通过活性区域交换的方法发现，MiCGTb的152位氨基酸是负责bis-C-糖基化反应的活性位点，而60/100/104位氨基酸则与bis-C-糖基化反应的活性有关。通过对上述四个活性位点进行理性设计，得到一系列具有催化活性高、底物谱宽不同催化功能的突变体酶，如MiCGT-E152L具有宽泛的糖基受体底物谱，MiCGTb-GANM等能够接受不同结构的α-D-和β-L-糖基供体以及能够催化结构多样的香豆素和酰基苯甲酮进行C-糖基化。这是目前报道的底物及反应杂泛性最强的bis-C-糖基转移酶。该研究不仅揭示了C-糖基转移酶催化bis-C-糖基化反应的关键活性位点，阐释了bis-C-糖基化的催化机制，而且通过人工设计改造构建了具体不同催化功能的C-糖基转移酶库，为活性糖苷类化合物的生物合成提供了新策略。同时，MiCGTb单酶法能选择性催化酰基间苯三酚2-O-糖苷合成3-C-糖苷。同时，我们组合了课题组前期发现的具杂泛性异戊烯基转移酶AtaPT,建立了同时对酰基间苯二酚类化合物进行C-糖基化和C-异戊烯基化方法，为该类化合物的分子创新与结构多样化、先导化合物发现奠定了基础。.本课题在实施过程中，已发表学术论文5篇（均标注基金资助），其中4篇为SCI收录论文，包括ACS Catalysis (IF: 12.221)、Organic Letters (IF: 6.555)、Chemistry-A European Journal (IF: 5.771)等本领域知名期刊，申请了1项专利。..