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 are inverting glycosyltransferases and 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 proposed to be involved in the final stages of the synthesis of highly bioactive triterpene saponins in the model legume Medicago truncatula. One of these UGTs, 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, firstly, 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.

Structure of M. truncatula UGT71G1

Enzyme interacts with UDP-glucose mainly through residues in the UGT signature PSPG motif (residue 339-382)

Substrate hederagenin was docked into the active site with the OH group at position 3 close to the NE2 atom of His22 (~3.2 Å), and the C1' atom of UDP-glucose (~3.8 Å).

Publications:
Li, L., Modolo, L.V., Escamilla-Trevino L.L., Achnine, L., Dixon, R.A., and Wang, X. Crystal structure of Medicago truncatula UGT85H2 - Insights into the structural basis of a multifunctional (iso)flavonoid glycosyltransferase. J. Mol. Biol. (2007), 370, 951-963.

Modolo, L.V., Blount, J.W., Achnine, L., Naoumkina, M.A., Wang, X., and Dixon, R.A. A functional genomics approach to (iso)flavonoid glycosylation in the model legume Medicago truncatula. Plant Molecular Biology (2007), 64, 499-518.

He, X., Wang, X., and Dixon, R.A. Mutational analysis of the Medicago glycosyltransferase UGT71G1 reveals residues that control regio-selectivity for (iso)flavonoid glycosylation. J. Biol. Chem. (2006), 281, 34441-34447.

Shao, H., He, X., Achnine, L., Blount, J.W., Dixon, R.A., and Wang, X. Crystal structures of a multifunctional triterpene/flavonoid glycosyltransferase from Medicago truncatula. Plant Cell (2005) Nov; 17(11): 3141-54.

Acknowledgment and Disclaimer:
This material is based upon work supported by the National Science Foundation under Grant No. 0416883. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.