Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. At this time, it is not possible to accurately predict putative GT activity from sequence alone; biochemical characterization is critical. A flavonol-specific 3-O-GT enzyme has been identified and cloned from the leaf tissues of grapefruit. The enzyme shows rigid substrate specificity and regiospecificity. F3GTs from grape and grapefruit were modeled against F7GTs from Crocus sativus and Scrutellaria biacalensis, and several non-conservative amino acid differences were identified that may impact regioselectivity. This research is designed to test the hypothesis that specific amino acid residues impart the regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-GT that were identified through homology modeling. Enzyme activity of the mutant F3-GT proteins will be analyzed for a possible change in glucosylation pattern. Other flavonoid classes will also be tested with the mutant enzymes to test for change in substrate specificity.

Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. At this time, it is not possible to accurately predict putative GT activity from sequence alone; biochemical characterization is critical. A flavonol-specific 3-O-GT enzyme has been identified and cloned from the leaf tissues of grapefruit. The enzyme shows rigid substrate specificity as well as regiospecificity. Several F3GT's characterized from other plants also had the ability to glucosylate anthocyanidins, however the grapefruit F3GT did not. This research is designed to test the hypothesis that specific amino acid residues impart the substrate specificity and regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-GT that were identified through homology modelling. Enzyme activity of the mutant F3-GT proteins will be tested with flavonols for a possible change in glucosylation pattern. Other flavonoid classes will also be tested with the mutant F3-GT enzyme to test for change in substrate specificity. The result from this study will add to our knowledge of GTs.