CRYSTALLIZATION AND ANALYSIS OF TAG EFFECTS USING A FLAVONOL SPECIFIC GLUCOSYLTRANSFERASE FROM GRAPEFRUIT
Location
Ballroom
Start Date
4-5-2018 8:00 AM
End Date
4-5-2018 12:00 PM
Poster Number
69
Name of Project's Faculty Sponsor
Cecilia McIntosh
Faculty Sponsor's Department
Biological Sciences
Type
Poster: Competitive
Project's Category
Natural Sciences
Abstract or Artist's Statement
Citrus and other fruits produce secondary metabolites that are synthesized, regulated, and modified by a class of enzymes called glucosyltransferases. This class of enzymes is of interest to this lab due to their unique structural and functional properties. Glucosides of flavonoids produced by glucosyltransferases are a critical part of plant metabolism and survival; many have health benefits when consumed. One such glucosyltransferase, found in Duncan grapefruit (Citrus paradisi), was identified, recombinantly expressed, and shown through biochemical characterization to exclusively glucosylate the flavonol class of flavonoids at the 3-OH position. The structural basis that accounts for a glucosyltransferase’s selectivity is not currently known, however great advances have been realized through the protein crystallization of 6 different secondary product glucosyltransferases. None of these show the same specificity exhibited by this flavonol-specific glucosyltransferase, CP3GT. The WT enzyme and two mutants have undergone site-directed mutagenesis to insert thrombin cleavage sites for removal of recombinant protein tags. The plasmid was transformed into yeast and protein was expressed through methanol induction. Cobalt column affinity chromatography was used to purify the protein. An aliquot of protein was treated with thrombin to remove tags and both tagged and native protein were assayed for activity with the flavonol quercetin. The data show that the reaction is linear for at least 15 minutes when 2ug of enzyme is used. Thus, kinetics assays will be conducted for 10 minutes. The presence of tags on the enzyme does not appear to impact activity with respect to the time course, however, more assays must be conducted to reliable confirm this with kinetic assays under different conditions. It is hypothesized that obtaining a crystal structure for this enzyme will illuminate the structural basis of its specificity. Additionally, it is hypothesized that a thrombin cleavage gene vector inserted for removal of purification tags will have no impact on enzyme activity or specificity.
CRYSTALLIZATION AND ANALYSIS OF TAG EFFECTS USING A FLAVONOL SPECIFIC GLUCOSYLTRANSFERASE FROM GRAPEFRUIT
Ballroom
Citrus and other fruits produce secondary metabolites that are synthesized, regulated, and modified by a class of enzymes called glucosyltransferases. This class of enzymes is of interest to this lab due to their unique structural and functional properties. Glucosides of flavonoids produced by glucosyltransferases are a critical part of plant metabolism and survival; many have health benefits when consumed. One such glucosyltransferase, found in Duncan grapefruit (Citrus paradisi), was identified, recombinantly expressed, and shown through biochemical characterization to exclusively glucosylate the flavonol class of flavonoids at the 3-OH position. The structural basis that accounts for a glucosyltransferase’s selectivity is not currently known, however great advances have been realized through the protein crystallization of 6 different secondary product glucosyltransferases. None of these show the same specificity exhibited by this flavonol-specific glucosyltransferase, CP3GT. The WT enzyme and two mutants have undergone site-directed mutagenesis to insert thrombin cleavage sites for removal of recombinant protein tags. The plasmid was transformed into yeast and protein was expressed through methanol induction. Cobalt column affinity chromatography was used to purify the protein. An aliquot of protein was treated with thrombin to remove tags and both tagged and native protein were assayed for activity with the flavonol quercetin. The data show that the reaction is linear for at least 15 minutes when 2ug of enzyme is used. Thus, kinetics assays will be conducted for 10 minutes. The presence of tags on the enzyme does not appear to impact activity with respect to the time course, however, more assays must be conducted to reliable confirm this with kinetic assays under different conditions. It is hypothesized that obtaining a crystal structure for this enzyme will illuminate the structural basis of its specificity. Additionally, it is hypothesized that a thrombin cleavage gene vector inserted for removal of purification tags will have no impact on enzyme activity or specificity.