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. Glucosyltransferases 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. The 382 position was of particular interest with arginine in C. paradisi and tryptophan in V. vinifera. This research was designed to test the hypothesis that a R382W mutation would result in altered substrate specificity. Site-directed mutagenesis was performed to form the R382W mutant Cp3OGT and the gene was transformed into yeast for protein 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. Docking analysis was performed using AutoDock software with both the wild type and R382W mutant protein with the substrates that showed interesting activity. The results of this study indicate that the point mutation of arginine to tryptophan at position 382 broadened substrate and regiospecificity to include flavanones.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:honors-1381 |
| Date | 01 August 2016 |
| Creators | King, Kathleen |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Undergraduate Honors Theses |
| Rights | Copyright by the authors., http://creativecommons.org/licenses/by-nc-nd/3.0/ |
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