Crystallization of a Unique Flavonol 3-O Glucosyltransferase found in Grapefruit
Location
Culp Room 303
Start Date
4-6-2022 9:00 AM
End Date
4-6-2022 10:00 AM
Faculty Sponsor’s Department
Biological Sciences
Name of Project's Faculty Sponsor
Cecilia McIntosh
Competition Type
Competitive
Type
Oral Presentation
Project's Category
Enzyme Catalysis
Abstract or Artist's Statement
Flavonoids are a specialized group of compounds produced by plants that give them greater adaptability to their environment and ultimately enhance their ability to survive. In plants, one function of flavonoids is to attract pollinators by their various flavor and scent profiles. They also protect the photosynthetic machinery from photo-oxidation. In humans, flavonoids have been shown to act as antioxidants, exhibit antimicrobial activity, and have shown potential as cancer treatments. In nature, flavonoids are most often found coupled with a sugar group (glucose, rhamnose, and others) which imparts stability and increases bioactivity. The process of adding a sugar (glycosylation) is catalyzed by a class of enzymes called glycosyltransferases (GT). One such enzyme found in grapefruit only glucosylates the flavonol class of flavonoids at the 3-OH position and is of interest due to its unique substrate and regio-specificity. Called Cp3GT (Citrus paradisi flavonol 3-O glucosyltransferase), this enzyme is similar in structure to other plant GT’s yet differs in the flavonoids it can glucosylate and where the glucose can be added. To date, the literature has not reported a structural mechanism for a flavonol specific 3-O glucosyltransferase’s unique catalytic activity. High-resolution structural imagery of enzymes, elucidated using X-ray crystallography, can be used to direct custom enzyme development to produce bioavailable natural products. Furthermore, structural research on enzymes with high specificity strengthens enzyme-ligand docking simulations, which are commonly used to test the binding affinity of potential pharmaceuticals. This research hypothesizes Cp3GT has structural features that confer its unique substrate and regiospecificity that are not revealed by homology modeling. This hypothesis will be tested using x-ray crystallography of purified Cp3GT protein bound to its preferred flavonol substrates. The gene for Cp3GT was transformed into Pichia pastoris and was recombinantly expressed using methanol induction. Cp3GT was purified to 80% purity using cobalt metal affinity chromatography. Cp3GT was subjected to additional purification measures using anion exchange chromatography with the goal of increasing purity to ≥95% for crystallization experiments. Purity analysis was conducted using SDS-PAGE (Coomassie/silver stain, western blot) and UV-Vis spectrophotometry. While initial results are promising, additional purification steps may be needed to achieve the purity necessary for crystallization.
Crystallization of a Unique Flavonol 3-O Glucosyltransferase found in Grapefruit
Culp Room 303
Flavonoids are a specialized group of compounds produced by plants that give them greater adaptability to their environment and ultimately enhance their ability to survive. In plants, one function of flavonoids is to attract pollinators by their various flavor and scent profiles. They also protect the photosynthetic machinery from photo-oxidation. In humans, flavonoids have been shown to act as antioxidants, exhibit antimicrobial activity, and have shown potential as cancer treatments. In nature, flavonoids are most often found coupled with a sugar group (glucose, rhamnose, and others) which imparts stability and increases bioactivity. The process of adding a sugar (glycosylation) is catalyzed by a class of enzymes called glycosyltransferases (GT). One such enzyme found in grapefruit only glucosylates the flavonol class of flavonoids at the 3-OH position and is of interest due to its unique substrate and regio-specificity. Called Cp3GT (Citrus paradisi flavonol 3-O glucosyltransferase), this enzyme is similar in structure to other plant GT’s yet differs in the flavonoids it can glucosylate and where the glucose can be added. To date, the literature has not reported a structural mechanism for a flavonol specific 3-O glucosyltransferase’s unique catalytic activity. High-resolution structural imagery of enzymes, elucidated using X-ray crystallography, can be used to direct custom enzyme development to produce bioavailable natural products. Furthermore, structural research on enzymes with high specificity strengthens enzyme-ligand docking simulations, which are commonly used to test the binding affinity of potential pharmaceuticals. This research hypothesizes Cp3GT has structural features that confer its unique substrate and regiospecificity that are not revealed by homology modeling. This hypothesis will be tested using x-ray crystallography of purified Cp3GT protein bound to its preferred flavonol substrates. The gene for Cp3GT was transformed into Pichia pastoris and was recombinantly expressed using methanol induction. Cp3GT was purified to 80% purity using cobalt metal affinity chromatography. Cp3GT was subjected to additional purification measures using anion exchange chromatography with the goal of increasing purity to ≥95% for crystallization experiments. Purity analysis was conducted using SDS-PAGE (Coomassie/silver stain, western blot) and UV-Vis spectrophotometry. While initial results are promising, additional purification steps may be needed to achieve the purity necessary for crystallization.