糖基转移酶UGT78H2的活性鉴定及在黑莓类黄酮代谢中的功能分析

Glycosylation of pant secondary metabolites is providing a direct mechanism affecting their activities, transportation and storage characteristics. Glycosyltransferases (GTs) are kinds of proteins catalysis this essential reaction. However, the mechanisms involved in sugar donor and acceptors selection remain unclear. We have identified a new GT family member UGT78H2, from the polyphenol enriched plants blackberry in our previous studies. From the results of sequence characteristics, phylogenetic inference, expression patterns, and the modeling and docking assays, it is certain that the protein takes part in glycosylation of small molecules other than anthocyanins. Deep further, there are two natural mutations in the 44 amino acids which constitute the conserved PSPG domain in plant UGTs. This domain was proved to act directly toward the substrates in the glycosylation reaction. It is therefore a good opportunity to test the recognition residues in the enzyme activity center. In this project, we are going to produce in vitro recombinant UGT78H2 as well as protein mutants (reverse mutations of the PSPG residues) via prokaryotic systems or eukaryotic bioreactors. The sugar donor and acceptors, and the end products of the UGT78H2 catalyzed reaction will be identified through HPLC/MS/NMR analysis. Enzyme kinetics of both the wild type and mutants will be determined and compared. Afterwards, subcellular localization of the protein in blackberry will be studied by using protoplasts transient expression. Over expression lines will be produced to identify the flavonoid alterations in different organs of the plants, hoping to unveil the in vivo function of UGT78H2. The accomplishment of this project will provide new evidence for understanding the reaction principles of UGTs, as well as bring new tools for side-direct molecular design of plant secondary metabolites.

植物次生代谢小分子化合物的糖基化直接影响其活性、转运和存储等特征。糖基转移酶GTs催化了这一重要反应，但其对糖基受体和糖基供体的选择机制尚未明晰。申请人前期从富含多酚的黑莓中获得了一个新的GT家族成员UGT78H2。结合序列特征、系统发育树、时序表达特征、蛋白同源建模和分子对接结果，该蛋白可能参与了非花青素类的类黄酮次生代谢终产物糖基化修饰。UGT78H2在UGT家族PSPG结构域与底物互作的保守氨基酸位点存在天然的突变，为研究糖基供体的识别机制提供了宝贵材料。本项目拟通过重组UGT78H2蛋白，分析其体外酶学反应活性；对差异位点进行回复突变，分析其底物和产物的变化情况；系统分析UGT78H2的亚细胞定位情况，超表达该基因对黑莓植株类黄酮产物的影响，阐释UGT78H2在黑莓类黄酮代谢中的功能。项目的实施将丰富UGTs底物选择识别机理，为代谢工程实现定向分子选育种提供新的工具。

糖基化修饰不仅可以影响植物次生代谢产物在植物细胞中的运输和存储进而影响其生物活性，同时也影响人类对产物的利用效率。尿苷二磷酸糖基转移酶（Uridine diphosphate glycosyltransferases，UGTs）是负责该修饰的多基因家族成员，其对糖基受体和供体底物的识别机制仍存在诸多问题。项目对前期分离的未知功能的黑莓UGT78H2进行重组表达，蛋白纯化，进一步对酶底物特异性和动力学参数进行了分析，对糖基转移酶PSPG结构域中两个重要氨基酸位点进行突变，分析了两个新位点的突变对酶促反应特性的影响。最后对该基因在黑莓不同组织中的表达特性，蛋白的亚细胞定位情况，基因启动子的时空表达特征进行了分析。结果表明：UGT78H2蛋白的糖基受体为槲皮素，识别的糖基受体为UDP-半乳糖和UDP-葡糖醛酸，催化生成的产物为槲皮素-3-O-半乳糖苷和槲皮素-7-O-β-葡糖苷；成功获得360天冬酰胺突变为脯氨酸，380位赖氨酸突变为天冬酰胺的μUGT78H2蛋白；突变后的蛋白识别的糖基受体仍然为槲皮素，但糖基供体除了UDP-半乳糖和UDP-葡糖醛酸外，还可以识别UDP-葡萄糖，增加的产物为槲皮素-4'-O-葡萄糖苷；两个蛋白识别的最适糖基供体分别为UDP-葡糖醛酸（Kcat/Km=0.91）和UDP-半乳糖（Kcat/Km=1.00）；UDP78H2主要表达在幼嫩组织中；蛋白于细胞质和细胞核中发挥功能；活性受赤霉素、乙烯、茉莉酸甲酯以及生长素等多种激素调控。课题揭示了通过对直接与糖基供体作用位点以外的氨基酸进行突变可以扩大UGT蛋白糖基选择范围的应用价值，也为黑莓育种提供了新的工具。