Flavonoids have multiple subclasses. A major subclass of flavonoids is flavonols. Flavonols are the most abundant subclass of flavonoids and are widely spread throughout nature. Flavonols are identified as having a hydroxyl group in the 3rd position of the C ring. The most prevalent modification to flavonols is glucosylation which adds glucose to an acceptor molecule. The flavonol specific 3-O glucosyltransferase (Cp3GT) enzyme from grapefruit (Citrus paradisi) is the topic of this research and specifically adds glucose to flavonols at the 3-OH position. The level of activity with Cp3GT and a flavonol varies depending on the flavonol structure. Since there is varying activity with Cp3GT, Cp3GT is an ideal model system for studying the structure/function relationship of Cp3GT site-directed mutants. Multiple mutants of Cp3GT were created by site directed mutagenesis. The mutant of study is S20G+T21S. As compared to the wild type Cp3GT, S20G+T21S has significantly higher activity with kaempferol, quercetin, dihydroquercetin, and naringenin. One of the more striking difference of S20G+T21S is its ability to add a glucose molecule to the 7-OH position of naringenin. Naringenin is a flavanone and indicates that S20G+T21S has a change in flavonoid class specificity as well as regiospecificity for the addition of glucose. The S20G+T21S mutant was first verified in E. coli via DNA sequencing. Next, S20G+T21S was transformed into Pichia pastoris by linearizing S20G+T21S DNA using Sac I, phenol chloroform purification, and electroporation. Transformation was verified by colony PCR and DNA sequencing. After verification, a time course analysis of expression conduction was completed. Optimal expression was concluded to be 24 hours and was verified by SDS-Page gel and western blot. In preparation for crystallization, S20G+T21S was purified using an IMAC infinity column. Purification was verified using a western blot, Coomassie blue, and silver stain. Progress on optimizing the crystallization conditions for S20G+T21S will be reported.

Flavonols are a major subclass of flavonoids and are considered the most abundant subclass of flavonoids. Flavonols are classified as having a hydroxyl group on the 3rd carbon of the C ring. The most prevalent modification to flavonols is glucosylation. The flavonol specific 3-O glucosyltransferase (Cp3GT) enzyme from grapefruit (Citrus paradisi) is the topic of this research and specifically adds glucose to flavonols at the 3-OH position. The level of activity varies depending on the flavonol structure. This makes Cp3GT an ideal model system for studying the structure/function relationship of Cp3GT using site-directed mutants. S20G+T21S is a mutant form of Cp3GT. As compared to the wild type Cp3GT, S20G+T21S has significantly higher activity with kaempferol, quercetin, and gossypetin. Another interesting difference of S20G+T21S is its ability to add glucose to the 7-OH position of the flavanone naringenin, thus showing a change in flavonoid class specificity as well as regiospecificity for position of glucose attachment. The S20G+T21S mutant was first made by site-directed mutagenesis and verified by DNA sequencing. The linearized pPICZa plasmid containing S20G+T21S was then transformed into Pichia pastoris via electroporation. Transformation was verified by colony PCR and DNA sequencing and a time course analysis of methanol-induced expression conducted to identify optimal expression. Optimal expression was identified at 24 hours and was verified by SDS-page gel and a western blot. S20G+T21S was purified by IMAC column in preparation for crystallization.