Structural and Functional Studies of Plant Natural Product Uridine Diphosphate Glycosyltransferases

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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.