Effect of Mutant P145T on the Enzyme Activity of Glucosyltransferase from Citrus paradisi

Flavonoids are the C-15 phenolic compounds containing two phenyl rings and a heterocyclic ring. The majority of the flavonoids accumulated in grapefruit are flavonol, flavanone, flavone, dihydroflavonol, and chalcone glycosides. Most flavonoids are present in glucosylated form and the glucosylation is mediated by a class of enzymes called glucosyltransferases that transfer glucose from a high energy sugar donor to the acceptor aglycone at a particular position. A clone encoding a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) from Citrus paradisi has been previously characterized in our lab. The study of structure and function of flavonoid GTs is an important aspect of our research that contributes to the synthesis of novel glucosides by changing the glucosylation patterns of GTs. Our study focuses on the structural and functional analysis of Cp-3-O-GT through site directed mutation and analysis of mutated enzyme in terms of substrate specificity and regiospecificity. Multiple sequence alignment and homology modeling was used to identify candidate areas for mutation. For this study, Cp-3-O-GT was modeled with a flavonoid 3- O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT – P145T mutant to test the hypothesis that that mutation of key amino acid residues (proline) in Cp-3-O-GT by position specific amino acids of VvGT (threonine) could alter substrate specificity or regiospecificity of Cp-3-O-GT. Initial screening results suggested that the mutant P145T glucosylates flavanones and flavones in addition to flavonols. This is significant because flavanones and flavonols do not contain a 3-OH group for glucosylation. HPLC was performed to identify the reaction products. Early results indicate that the P145T mutant glucosylates naringenin at 7-OH position forming naringenin-7-O-glucoside and this is being confirmed. Product identification with other substrates is also being conducted. Results are being used to revisit and refine the structure model