Glucosinolates are a diverse class of natural compounds synthesized by plants and vegetables of the crucifer family. The functions of glucosinolates in plants are largely unknown. However, their hydrolytic breakdown products exert a variety of biological activities in plants, animals, and humans, which range from the participation in plant defense against pathogens and herbivores to the prevention of cancers. Although substantial progress has been made in understanding glucosinolate metabolism, many of the genes involved in glucosinolate biosynthesis and its regulation remain to be identified or warrant functional characterization. Glucosinolates are synthesized from amino acids in three major stages: (i) side-chain elongation of the parental amino acids, (ii) formation of the glycone moiety that is common to all glucosinolates, and (iii) modification of the (elongated) amino acid side-chain. The second stage involves transfer of a glucose molecule (glucosylation) to intermediate compounds known as thiohydroximates. Based on biochemical studies by others in Brassica, a gene in Arabidopsis has been identified by us that encodes a putative thiohydroximate glucosyltransferase (UGT74B1). Preliminary data point to a biochemical and biological role for UGT74B1 in glucosinolate biosynthesis. Furthermore, the results suggest that additional enzymes exist in Arabidopsis that catalyze glucosylation of thiohydroximate intermediates. Therefore, the following objectives are proposed: (i) to thoroughly characterize the catalytic activity and substrate specificity of UGT74B1; (ii) to extend our studies to the discovery of additional thiohydroximate glucosyltransferases in Arabidopsis; (iii) to select the most potent enzymes for a comparative biochemical analysis of their kinetic properties and substrate specificities; and (iv) to study the function of the corresponding genes in glucosinolate biosynthesis as well as during plant development by various genetic approaches. The proposed research will lead to a more comprehensive understanding of the glucosinolate pathway and will contribute to the functional characterization of plant glucosyltransferases, which have largely unknown but diverse functions in primary and secondary metabolism. In the broader perspective, the availability of characterized genes with functions in glucosinolate biosynthesis and its regulation will enable rational metabolic engineering of plants for altered glucosinolate profiles, with the prospect to reduce antinutritional glucosinolates in Brassica seeds for animal feed, to modify glucosinolate composition for crop protection, or to design functional foods with optimal content of health-beneficial glucosinolates in cancer prevention strategies. In addition, this highly collaborative project will provide an excellent opportunity for continued high-level research training in broad and interdisciplinary areas of contemporary molecular biology and biochemistry, which will target postdoctoral researchers, undergraduate students and motivated high school students of diverse backgrounds with interests in experimental biology.
