Structure-Function Investigations of Site-Directed Mutants of Citrus paradisi Flavonol-Specific 3 O Glucosyltransferase (Cp3OGT) – Impact of Mutations of Serine, Histidine, and Glutamine
Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.
Sathanantham, Preethi; Shivakumar, Devaiah P.; and McIntosh, Cecelia A.. 2015. Structure-Function Investigations of Site-Directed Mutants of Citrus paradisi Flavonol-Specific 3 O Glucosyltransferase (Cp3OGT) – Impact of Mutations of Serine, Histidine, and Glutamine. Poster presentation. Annual Meeting of the Phytochemical Society of North America, Champaign, IL. http://conferences.illinois.edu/psna/documents/PSNA_2015_Full_Program.pdf