Project Title

CRYSTALLIZATION AND ANALYSIS OF TAG EFFECTS USING A FLAVONOL SPECIFIC GLUCOSYLTRANSFERASE FROM GRAPEFRUIT

Authors' Affiliations

East Tennessee State University, Department of Biological Sciences, Johnson City, TN 37601, USA

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

Classification of First Author

Graduate Student-Master’s

Project's Category

Natural Sciences

Abstract Text

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.

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Apr 5th, 8:00 AM Apr 5th, 12:00 PM

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.