Structural and Functional Studies of Plant Natural Product Uridine Diphosphate Glycosyltransferases

糖苷键的酶促形成在数量上是地球上最重要的反应，并且由酶的超家族（糖基转移酶（GT））催化，所述酶已被分类为超过60个家族。 这个项目的重点是家族1酶，尿苷二磷酸（UDP）糖基转移酶（UGT）。UGT将UDP活化的糖部分转移到特定的受体分子。 在植物中，存在可能参与次级代谢物的糖基化的组1 UGT的大家族。几个UGT已被假定参与高生物活性的三萜皂苷在模式豆科植物蒺藜苜蓿的合成的最后阶段。其中之一，GT 029 H，糖基化皂苷糖苷配基medicagenic acid。本项目的主要目标是了解家族1 UGT催化糖基化反应的分子机制。 目的是确定蒺藜苜蓿中两种三萜UGT与糖供体UDP葡萄糖、受体底物或/和抑制剂复合物的晶体结构，以揭示酶与供体和受体之间的相互作用，并阐明酶催化机制。通过对UGT突变体的研究，可以更全面地探讨糖基化反应中关键残基的作用机制。通过探索植物UGT超家族更广泛的横截面之间的结构-功能关系，使用X射线晶体学，同源建模和酶/生化分析，该项目将开发UGT底物特异性的预测模型。在上述领域的成功将提供出发点，为合理操纵的底物和产品的具体个别UGT，与植物的代谢工程的影响，增加生产有价值的，生物活性的次级代谢产物。 更广泛的影响：通过结合涉及基础植物生物学，蛋白质生物化学，化学和生物物理学的方法，该项目将提供一个真正的跨学科博士后培训的工具。将实施一项培训本科生将结构生物学应用于植物生物化学和生物技术的方案，并将通过实践和网上学习机会扩大对当地高中生的外展活动。

The enzymatic formation of glycosidic bonds is quantitatively the most significant reaction on earth and is catalyzed by a superfamily of enzymes, the glycosyltransferases (GTs), which have been classified into over 60 families. This project focuses on the family 1 enzymes, the uridine diphosphate (UDP) glycosyltransferases (UGTs). UGTs transfer UDP-activated sugar moieties to specific acceptor molecules. In plants, there are large families of group 1 UGTs that may be involved in the glycosylation of secondary metabolites. Several UGTs have been postulated to be involved in the final stages of the synthesis of highly bioactive triterpene saponins in the model legume Medicago truncatula. One of these, GT029H, glycosylates the saponin aglycone medicagenic acid. The major goal of this project is to understand the molecular mechanisms of glycosylation reactions catalyzed by family 1 UGTs. The objectives are to determine the crystal structures of two triterpene UGTs from Medicago truncatula in complexes with the sugar donor UDP glucose, acceptor substrates or/and inhibitors, in order to reveal the interactions between the enzymes and the donor and acceptor, and to elucidate the enzyme catalytic mechanism. A study of mutants of UGTs will fully explore the mechanism and roles of key residues for the glycosylation reaction. By exploring structure-function relations among a broader cross-section of the plant UGT superfamily, using x-ray crystallography, homology modeling, and enzymatic/biochemical analysis, the project will develop predictive models for UGT substrate specificity. Success in the above areas will provide starting points for the rational manipulation of the substrate and product specificities of individual UGTs, with implications for the metabolic engineering of plants for increased production of valuable, bioactive secondary metabolites. Broader Impact: By combining approaches involving basic plant biology, protein biochemistry, chemistry and biophysics, this project will provide a vehicle for truly interdisciplinary postdoctoral training. A program for training undergraduates in the application of structural biology to plant biochemistry and biotechnology will be put in place, and outreach activities to local high school students through hands-on and web-based learning opportunities will be extended.