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Presentation
Structure and Functional Analysis of Glucosyltransferase from Citrus paradisi
29th Annual Appalachian Student Research Forum
  • Shivakumar P. Devaiah, East Tennessee State University
  • Cheng Zhang, East Tennessee State University
  • Cecelia A. McIntosh, East Tennessee State University
Document Type
Presentation
Publication Date
4-2-2014
Description
Glucosyltransferases (GTs) are enzymes that expedite the incorporation of UDP-activated glucose to a corresponding acceptor molecule. This enzymatic reaction stabilizes structures and affects solubility, transport, and bioavailability of flavonoids for other metabolic processes. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant flavonol-3-O-glucosyltransferase (F-3-O-GT) proteins using site-directed mutagenesis and testing the effect of each mutation on substrate specificity, regiospecificity and kinetic properties of the enzyme. Mutations were selected on the basis of sequence similarity between grapefruit F-3-O-GT, an uncharacterized GT gene in blood orange (98%), and grape F3GT (82%). Grapefruit F-3-O-GT prefers flavonol as a substrate whereas the blood orange sequence is annotated to be a flavonoid 3GT and the grape GTs could glucosylate both flavonols and anthocyanidins. Mutants of F-3-O-GT were generated by substituting L41M, N242K, E296K and N242K+E296K and proteins were expressed in Pichia pastoris using the pPICZA vector. Analysis of these mF-3-O-GTs showed that all of them preferred flavonols over flavanone, flavone, isoflavones, or anthocyanidin substrates and showed decrease in enzyme activity of 16 to 51% relative to the wild type F-3-O-GT.
Location
Johnson City, TN
Citation Information
Shivakumar P. Devaiah, Cheng Zhang and Cecelia A. McIntosh. "Structure and Functional Analysis of Glucosyltransferase from Citrus paradisi" 29th Annual Appalachian Student Research Forum (2014)
Available at: http://works.bepress.com/cecilia-mcintosh/64/