Structural and Functional Analysis of Grapefruit C-3OGT Mutant P145T
Flavonoids are a class of secondary metabolites, the majority of which are present in glucosylated form. Glucosyltransferases are the enzymes that mediate glucosylation by transferring glucose from a high energy sugar donor to the acceptor substrates. Our study focuses on the structural and functional analysis of a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) clone from Citrus paradisi that has been characterized previously in our lab. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT- P145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-OGT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavonols do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at 7-OH position forming prunin. Product identification with other substrates is in progress. Results are being used to revisit and refine the structure model. Structural and functional analysis of flavonoid GTs may contribute to custom design of GTs for the synthesis of novel glucosides by changing glucosylation patterns.
Kandel, Sangam; Khaja, Sarah; Shivakumar, Devaiah P.; and McIntosh, Cecelia A.. 2015. Structural and Functional Analysis of Grapefruit C-3OGT Mutant P145T. Poster presentation. Annual Meeting of the Phytochemical Society of North America, Champaign, IL. http://conferences.illinois.edu/psna/documents/PSNA_2015_Full_Program.pdf