Flavonol Specific 3-O Glucosyltransferase (Cp3GT) Mutant S20G+T21S: Enzyme Structure and Function
Honors in Biology
Date of Award
Dr. Cecilia A. McIntosh
Thesis Professor Department
<--College of Arts and Sciences-->
Dr. Dhirendra Kumar and Dr. Ranjan Chakraborty
Flavonols are a major subclass of flavonoids and are considered the most abundant subclass of flavonoids. Flavonols are classified as having a hydroxyl group on the 3rd carbon of the C ring. The most prevalent modification to flavonols is glucosylation. The flavonol specific 3-O glucosyltransferase (Cp3GT) enzyme from grapefruit (Citrus paradisi) is the topic of this research and specifically adds glucose to flavonols at the 3-OH position. The level of activity varies depending on the flavonol structure. This makes Cp3GT an ideal model system for studying the structure/function relationship of Cp3GT using site-directed mutants. S20G+T21S is a mutant form of Cp3GT. As compared to the wild type Cp3GT, S20G+T21S has significantly higher activity with kaempferol, quercetin, and gossypetin. Another interesting difference of S20G+T21S is its ability to add glucose to the 7-OH position of the flavanone naringenin, thus showing a change in flavonoid class specificity as well as regiospecificity for position of glucose attachment.
The S20G+T21S mutant was first made by site-directed mutagenesis and verified by DNA sequencing. The linearized pPICZa plasmid containing S20G+T21S was then transformed into Pichia pastoris via electroporation. Transformation was verified by colony PCR and DNA sequencing and a time course analysis of methanol-induced expression conducted to identify optimal expression. Optimal expression was identified at 24 hours and was verified by SDS-page gel and a western blot. S20G+T21S was purified by IMAC column in preparation for crystallization.
East Tennessee State University
Honors Thesis - Withheld
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Fobare, Hayden, "Flavonol Specific 3-O Glucosyltransferase (Cp3GT) Mutant S20G+T21S: Enzyme Structure and Function" (2020). Undergraduate Honors Theses. Paper 720. https://dc.etsu.edu/honors/720
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