Glucosyltransferases (GTs) are the important group of enzymes which facilitates the incorporation of UDPactivated glucose to a corresponding acceptor molecule through glucosylation. Glucosylation is a common alteration reaction in plant metabolism and is regularly associated with the production of secondary metabolites. Glucosylation serves a number of roles within metabolism including: stabilizing structures, affecting solubility, transport, and regulating the bioavailability of the compounds for other metabolic processes. GTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase (PSPG) box, which has been demonstrated to include the UDP-sugar binding moiety. Among the secondary metabolites, flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant Flavonol- 3-O- Glucosyltransferases proteins using site-directed mutational analysis and testing the effect of each mutation on substrate specificity and kinetic properties of the enzyme.

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. A flavonol-specific 3-O-GT enzyme has been identified and cloned from leaf tissues of grapefruit. The enzyme shows rigid substrate specificity and regiospecificity. F3-O-GTs from grape (Vitis vinifera) and grapefruit (Citrus paradisi) were modeled against F7-O-GTs 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-O-GT that were identified through homology modeling. Analyses of the enzyme activity of the mutant F3-O-GT proteins revealed that the single point mutations of serine 20 to leucine (S20L) and proline 297 to phenylalanine (P297F) rendered the recombinant enzyme inactive with flavonol substrates. Mutation of glycine 392 to glutamate (G392E) was active at 80% relative to the wild type. The mutant enzyme also did not show broadened acceptor specificity as it also favored flavonols as the preferred acceptor substrate. The glucosylation products of the active mutant enzyme will be analyzed to determine if this resulted in a change in regiospecificity.