Date of Award
Thesis Professor Department
Ranjan Chakraborty,Dhirendra Kumar
Plants produce a vast array of secondary metabolites. A group of phenolic compounds, the flavonoids, are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have been found to possess anti-inflammatory, anti-cancer, and antioxidant properties. Flavonoid’s ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases (GT). Citrus paradisi contains a glucosyltransferase that is specific for adding glucose to the 3-O position of flavonols (Cp-F3-O-GT). To further understand the reactions it catalyzes, Cp-F3-O-GT structure was modeled against an anthocyanidin/flavonol 3-O-GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then generated using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline. Biochemical characterization of the mutant D344P protein was performed in order to determine whether the mutation has an effect on the substrate specificity of Cp3-O-GT. An initial quick-screening assay using radioactive UDP-glucose as a sugar donor suggested there may have been an expansion of substrate acceptance. The time course assays did not support observation. Additionally, results show that D344P protein has decreased activity with flavonols as compared to the wild-type Cp3-O-GT. with no expansion of substrate specificity. Homology models supported experimental results.
East Tennessee State University
Honors Thesis - Open Access
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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
Spaulding, Nathan R., "Determination of the Substrate Specificity of Citrus paradisi Flavonol Specific 3-O-Glucosyltransferase Mutant D344P" (2017). Undergraduate Honors Theses. Paper 372. http://dc.etsu.edu/honors/372
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