The Effect of R382W Mutation on C. paradisi Flavonol-Specific 3-O-Glucosyltransferase

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Flavonoids are a class of plant metabolites with C6-C3-C6 structure responsible for many biological functions, including coloration and defense. Citrus paradisi, grapefruit, contains a wide variety of flavonoids which are grouped by the extent of modification, examples of which are flavonols, flavones, and flavanones. A major modification is the addition of glucose by glucosyltransferases (GTs) to stabilize the structure and provide ease of transport. This process can be highly substrate and regiospecific. With Cp3OGT, glucose is added at the 3-hydroxy position. This 3GT only accepts flavonols as its substrate; however, a Vitis vinifera (grape) 3-GT can accept both flavonols and anthocyanidins. Homology modeling using the crystallized structure of the V. vinifera GT predicted sites of amino acids that could influence substrate binding site. The 382 position was of particular interest with arginine in C. paradisi and tryptophan in V. vinifera. This change is hypothesized to cause a shift in substrate specificity of the Cp3OGT to accept anthocyanidins as well as flavonols. Site-directed mutagenesis was performed to form the R382W mutant Cp3OGT and transformed into yeast for expression. Western blot determined the optimal protein induction period for the cells, after which the cells were broken to extract the recombinant mutant protein. Purification of the R382W 3GT allowed for enzyme analysis to be performed by measuring the incorporation of radioactive glucose into the reaction product. HPLC will be used to identify reaction products. An enzyme kinetics study will show the extent of any biochemical change in function as a result of this mutation; results will then be incorporated into a refined protein model.


Champaign, IL

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