Project Title

Flavonol Specific 3-O Glucosyltransferase (Cp3GT) Mutant S20G+T21S: Enzyme Structure and Function

Authors' Affiliations

Hayden Fobare, Department of Biological Sciences, East Tennessee State University, Johnson City, TN

Location

Culp Room 210

Start Date

4-6-2022 9:00 AM

End Date

4-6-2022 9:15 AM

Faculty Sponsor’s Department

Biological Sciences

Name of Project's Faculty Sponsor

Cecilia McIntosh

Additional Sponsors

Ranjan Chakraborty and Dhirendra Kumar

Classification of First Author

Undergraduate Student

Competition Type

Non-Competitive

Type

Boland Symposium

Project's Category

Biochemistry

Abstract or Artist's Statement

Flavonoids have multiple subclasses. A major subclass of flavonoids is flavonols. Flavonols are the most abundant subclass of flavonoids and are widely spread throughout nature. Flavonols are identified as having a hydroxyl group in the 3rd position of the C ring. The most prevalent modification to flavonols is glucosylation which adds glucose to an acceptor molecule. 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 with Cp3GT and a flavonol varies depending on the flavonol structure. Since there is varying activity with Cp3GT, Cp3GT is an ideal model system for studying the structure/function relationship of Cp3GT site-directed mutants. Multiple mutants of Cp3GT were created by site directed mutagenesis. The mutant of study is S20G+T21S. As compared to the wild type Cp3GT, S20G+T21S has significantly higher activity with kaempferol, quercetin, dihydroquercetin, and naringenin. One of the more striking difference of S20G+T21S is its ability to add a glucose molecule to the 7-OH position of naringenin. Naringenin is a flavanone and indicates that S20G+T21S has a change in flavonoid class specificity as well as regiospecificity for the addition of glucose.

The S20G+T21S mutant was first verified in E. coli via DNA sequencing. Next, S20G+T21S was transformed into Pichia pastoris by linearizing S20G+T21S DNA using Sac I, phenol chloroform purification, and electroporation. Transformation was verified by colony PCR and DNA sequencing. After verification, a time course analysis of expression conduction was completed. Optimal expression was concluded to be 24 hours and was verified by SDS-Page gel and western blot. In preparation for crystallization, S20G+T21S was purified using an IMAC infinity column. Purification was verified using a western blot, Coomassie blue, and silver stain. Progress on optimizing the crystallization conditions for S20G+T21S will be reported.

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Apr 6th, 9:00 AM Apr 6th, 9:15 AM

Flavonol Specific 3-O Glucosyltransferase (Cp3GT) Mutant S20G+T21S: Enzyme Structure and Function

Culp Room 210

Flavonoids have multiple subclasses. A major subclass of flavonoids is flavonols. Flavonols are the most abundant subclass of flavonoids and are widely spread throughout nature. Flavonols are identified as having a hydroxyl group in the 3rd position of the C ring. The most prevalent modification to flavonols is glucosylation which adds glucose to an acceptor molecule. 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 with Cp3GT and a flavonol varies depending on the flavonol structure. Since there is varying activity with Cp3GT, Cp3GT is an ideal model system for studying the structure/function relationship of Cp3GT site-directed mutants. Multiple mutants of Cp3GT were created by site directed mutagenesis. The mutant of study is S20G+T21S. As compared to the wild type Cp3GT, S20G+T21S has significantly higher activity with kaempferol, quercetin, dihydroquercetin, and naringenin. One of the more striking difference of S20G+T21S is its ability to add a glucose molecule to the 7-OH position of naringenin. Naringenin is a flavanone and indicates that S20G+T21S has a change in flavonoid class specificity as well as regiospecificity for the addition of glucose.

The S20G+T21S mutant was first verified in E. coli via DNA sequencing. Next, S20G+T21S was transformed into Pichia pastoris by linearizing S20G+T21S DNA using Sac I, phenol chloroform purification, and electroporation. Transformation was verified by colony PCR and DNA sequencing. After verification, a time course analysis of expression conduction was completed. Optimal expression was concluded to be 24 hours and was verified by SDS-Page gel and western blot. In preparation for crystallization, S20G+T21S was purified using an IMAC infinity column. Purification was verified using a western blot, Coomassie blue, and silver stain. Progress on optimizing the crystallization conditions for S20G+T21S will be reported.