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Article
Mutational Analysis of Substrate Specificity in a Citrus paradisi Flavonol 3-O-glucosyltransferase
Journal of Plant Biochemistry and Biotechnology (2017)
  • Shivakumar P. Devaiah, East Tennessee State University
  • Benjamin M. Tolliver, East Tennessee State University
  • Cheng Zhang, East Tennessee State University
  • Daniel K. Owens, University of Hawaii
  • Cecilia McIntosh, East Tennessee State University
Abstract
Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis vinifera anthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.
Keywords
  • citrus paradisi,
  • citrus sinensis,
  • flavonoid,
  • glucosyltransferase,
  • site-directed mutagenesis,
  • substrate specificity
Publication Date
June 8, 2017
DOI
10.1007/s13562-017-0411-0
Citation Information
Shivakumar P. Devaiah, Benjamin M. Tolliver, Cheng Zhang, Daniel K. Owens, et al.. "Mutational Analysis of Substrate Specificity in a Citrus paradisi Flavonol 3-O-glucosyltransferase" Journal of Plant Biochemistry and Biotechnology (2017) ISSN: 0974-1275
Available at: http://works.bepress.com/cecilia-mcintosh/24/