Crystallization of a Flavonol-Specific 3-O-Glucosyltrasnferase found in Citrus paradisi
Citrus and other fruits produce secondary metabolites that are synthesized, regulated, and modified in part by a class of enzymes called glycosyltransferases. This class of enzymes is of substantial interest to this lab due to their unique structural and functional properties. Glycosides of flavonoids produced by glycosyltransferases have emerged in recent years as a critical part of plant metabolism, thus impacting every aspect of their growth, cultivation, production, and utilization. One such glycosyltransferase, found in Duncan Grapefruits (Citrus paradisi), was previously identified, recombinantly expressed, and shown through biochemical characterization to exclusively glycosylate the flavonol class of flavonoids. The structural basis that accounts for a glycosyltransferase's selectivity has been determined by protein crystallization in other labs, yet no structural basis currently exists for the specificity exhibited by this flavonol-specific glycosyltransferase. Currently, the WT enzyme and two mutants were expressed in E. coli, where they underwent site-directed mutagenesis to insert thrombin cleavage tags for removal of protein purification vectors, with the goal of transforming into yeast for adequate protein production. Subsequent purification and crystallization screens will allow for formation and acquisition of glycosyltransferase crystals, whose x-ray diffraction patterns will be decoded, thus revealing the enzyme's complete structure. We hypothesize that obtaining a crystal structure for this enzyme will illuminate the structural basis of its specificity. Additionally, we hypothesize that a thrombin- cleavage gene vector inserted for removal of purification tags will have no impact on enzyme activity or specificity.
Johnson City, TN
Birchfield, Aaron; and McIntosh, Cecelia A.. 2017. Crystallization of a Flavonol-Specific 3-O-Glucosyltrasnferase found in Citrus paradisi. Poster presentation. 2017 Appalachian Student Research Forum, Johnson City, TN. http://www.etsu.edu/studentresearch/2017/documents/2017_asrf_programbook.pdf