Flavonoids are an important group of secondary metabolites found in plants and have a wide variety of properties. Some play a role in fl ower pigmentation, while others have antimicrobial properties. Glucosylation is an important modifi cation of fl avonoids and is mediated by glucosyltransferases. In this process, the enzyme transfers glucose from UDP-glucose to a specifi c position on the fl avonoid. Previous study from the lab characterized a glucosyltransferase from C. paradisi that is fl avonol specifi c. In this study an attempt has been made to study the structure and function of this fl avonol specifi c glucosyltransferase using site directed mutagenesis. The glutamine residue at position 87 of the Cp-3-O-GT enzyme was changed to isoleucine, the analogous residue in the 3-O-glucosyltransferase of Clitoria ternatea. Similarly, the histidine at position 154 was changed to tyrosine. We hypothesize that these mutations will change substrate specifi city. The glutamate at position 88 was changed to an aspartic acid. We hypothesize that this will change the regiospecifi city of the enzyme, as aspartic acid is the analogous residue found in some 7-O-glucosyltransferases. Finally, we introduced a double mutation with glutamine 87 becoming isoleucine and glutamate 88 becoming aspartic acid, with the hypothesis that both regiospecifi city and substrate specifi city will be changed.

Glucosyltransferases (GTs) are enzymes that expedite the incorporation of UDP-activated glucose to a corresponding acceptor molecule. This enzymatic reaction stabilizes structures and affects solubility, transport, and bioavailability of flavonoids for other metabolic processes. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant flavonol-3-O-glucosyltransferase (F-3-O-GT) proteins using site directed mutagenesis and testing the effect of each mutation on substrate specificity, regiospecificity and kinetic properties of the enzyme. Mutations were selected on the basis of sequence similarity between grapefruit F-3- O-GT, an uncharacterized GT gene in blood orange (98%), and grape F3GT (82%). Grapefruit F-3-O-GT prefers flavonol as a substrate whereas the blood orange sequence is annotated to be a flavonoid 3GT and the grape GTs could glucosylate both flavonols and anthocyanidins. Mutants of F-3-O-GT were generated by substituting N242K, E296K and N242K+E296K and proteins were expressed in Pichia pastoris using the pPICZA vector. Analysis of these mF-3-O-GTs showed that all of them preferred flavonols over flavanone, flavone, isoflavones, or anthocyanidin substrates and showed decrease in enzyme activity of 16 to 51% relative to the wild type F-3- O-GT.

Plants produce a vast array of secondary metabolites. The phenolic compounds flavonoids are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have also been found to possess anti-inflammatory, anti-cancer, and anti-oxidant properties. Flavonoids ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases. Citrus paradisi contains a glucosyltransferase that is specific to the 3-O position of flavonols. To further understand the reactions it catalyzes, Cp3-O-GT structure was modeled against an anthocyanidin/flavonol 3 GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then created using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline based off of the sequence comparison of the original enzymes. Biochemically characterizing the mutant D344P protein will determine whether the mutation has an effect on the substrate specificity of Cp3-O-GT. An initial quickscreening assay using radioactive UDP-glucose as a sugar donor suggested there may have been expansion of substrate acceptance. Confirming time course assays did not support this. Additionally, results of these assays show that D344P protein has decreased activity with flavonols as compared to wild type Cp3-O-GT. with no expansion of substrate specificity. Models suggest that a change in protein conformation has resulted in decreased activity.