Structural and Functional Studies of Plant Natural Product Uridine Diphosphate Glycosyltransferases

糖苷键的酶促形成是地球上最重要的定量反应，由一个超家族的酶催化，糖基转移酶（GTs），已被划分为60多个家族。本项目主要研究1家族酶，尿苷二磷酸（UDP）糖基转移酶（UGTs）。ugt将udp活化的糖部分转移到特定的受体分子上。在植物中，有一大家族的1族ugt可能参与次生代谢产物的糖基化。几种ugt被认为参与了模式豆科植物紫花苜蓿高生物活性三萜皂苷合成的最后阶段。其中之一，GT029H，将皂素苷元药原酸糖基化。本项目的主要目的是了解家族1 UGTs催化糖基化反应的分子机制。目的是测定两种三萜类ugt与糖供体UDP葡萄糖、受体底物或/和抑制剂配合物的晶体结构，以揭示酶与供体和受体的相互作用，并阐明酶的催化机制。对ugt突变体的研究将充分探索糖基化反应关键残基的机制和作用。通过探索更广泛的植物UGT超家族的结构-功能关系，使用x射线晶体学，同源性建模和酶/生化分析，该项目将开发UGT底物特异性的预测模型。上述领域的成功将为合理操纵单个ugt的底物和产物特异性提供起点，对植物代谢工程增加有价值的、生物活性的次生代谢物的生产具有重要意义。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，该项目将为真正跨学科的博士后培养提供一个载体。将实施结构生物学在植物生物化学和生物技术中的应用的本科生培训计划，并将通过实践和网络学习机会扩大对当地高中生的推广活动。

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.