Crystallization of a Flavonol-Specific 3-O Glucosyltransferase and Site-Directed Mutants from Grapefruit

Authors' Affiliations

1 and 2: Department of Biological Sciences, East Tennessee State University, Johnson City, TN 3761

Location

Ballroom

Start Date

4-12-2019 9:00 AM

End Date

4-12-2019 2:30 PM

Poster Number

2

Faculty Sponsor’s Department

Biological Sciences

Name of Project's Faculty Sponsor

Dr. Cecilia McIntosh

Classification of First Author

Graduate Student-Doctoral

Type

Poster: Competitive

Project's Category

Molecular Biology, Protein Structure, Biochemistry

Abstract or Artist's Statement

Citrus fruits are some of the most widely consumed fruits in the world and contain significant levels of flavonoids, a category of plant secondary metabolites which control taste, color, plant defense, and overall marketability. In citrus and other plants, flavonoids are found in their glucosylated form. Glucosyltransferases (GT’s) are enzymes that add glucose to secondary metabolites like flavonoids. They make up a diverse class of enzymes ubiquitous throughout the plant and animal kingdoms. While many GT’s have been identified, they vary greatly in their structural identity, and their chemical properties make it such that only a small percentage of existing GT’s have been functionally characterized. Research on GT structure function relationships strengthens the reliability of genomic databases and makes significant contributions to the field of enzyme biotechnology. Bioenergy research and custom enzyme synthesis rely on GT structural data, making this research critical to the success of many promising current and future projects. A GT was isolated from grapefruit and was shown to glucosylate the flavonol class of flavonoids at the 3-OH position, called CP3GT. Subsequent analysis showed there are specific arrangements of amino-acids inside the catalytic cleft of CP3GT that likely account for its specificity with flavonols. These interactions are not fully understood and make CP3GT an excellent model for elucidating unique structure function relationships of a GT enzyme. X-ray crystallography is one of the best methods for structure determination that allows a 3D image of the protein in question to be resolved at the molecular level. This method has vast potential for advancing plant enzymology, yet to date only 6 plant glucosyltransferases have had their crystal structures solved. The structural similarities and complementary specificities that CP3GT shares with these crystallized GT’s make CP3GT an excellent candidate for crystallization. This research hypothesizes that there are unique structural features that give CP3GT its specificity, and that these features can be elucidated using x-ray crystallography. Wild type CP3GT and 3 recently characterized mutants are being prepared for crystallization. The crystallization of 3 CP3GT mutants in addition to wild type will compliment structure/function analysis by providing insight into how structural modifications can alter enzyme function. It is recommended that protein be in its native form for crystallization, thus a thrombin-cleavage site was inserted into WT CP3GT and 3 mutants to remove tags following purification. Some studies have suggested that the presence of tags alters enzyme activity, thus this presented the opportunity to test the effect of tags by assaying both native and tagged enzyme. Initial results showed that WT CP3GT treated with thrombin retained 70 percent activity after a 2-hour treatment at 4o C. Additional assays will be conducted to fully determine tag effects and will run concurrently with crystallization experiments

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Apr 12th, 9:00 AM Apr 12th, 2:30 PM

Crystallization of a Flavonol-Specific 3-O Glucosyltransferase and Site-Directed Mutants from Grapefruit

Ballroom

Citrus fruits are some of the most widely consumed fruits in the world and contain significant levels of flavonoids, a category of plant secondary metabolites which control taste, color, plant defense, and overall marketability. In citrus and other plants, flavonoids are found in their glucosylated form. Glucosyltransferases (GT’s) are enzymes that add glucose to secondary metabolites like flavonoids. They make up a diverse class of enzymes ubiquitous throughout the plant and animal kingdoms. While many GT’s have been identified, they vary greatly in their structural identity, and their chemical properties make it such that only a small percentage of existing GT’s have been functionally characterized. Research on GT structure function relationships strengthens the reliability of genomic databases and makes significant contributions to the field of enzyme biotechnology. Bioenergy research and custom enzyme synthesis rely on GT structural data, making this research critical to the success of many promising current and future projects. A GT was isolated from grapefruit and was shown to glucosylate the flavonol class of flavonoids at the 3-OH position, called CP3GT. Subsequent analysis showed there are specific arrangements of amino-acids inside the catalytic cleft of CP3GT that likely account for its specificity with flavonols. These interactions are not fully understood and make CP3GT an excellent model for elucidating unique structure function relationships of a GT enzyme. X-ray crystallography is one of the best methods for structure determination that allows a 3D image of the protein in question to be resolved at the molecular level. This method has vast potential for advancing plant enzymology, yet to date only 6 plant glucosyltransferases have had their crystal structures solved. The structural similarities and complementary specificities that CP3GT shares with these crystallized GT’s make CP3GT an excellent candidate for crystallization. This research hypothesizes that there are unique structural features that give CP3GT its specificity, and that these features can be elucidated using x-ray crystallography. Wild type CP3GT and 3 recently characterized mutants are being prepared for crystallization. The crystallization of 3 CP3GT mutants in addition to wild type will compliment structure/function analysis by providing insight into how structural modifications can alter enzyme function. It is recommended that protein be in its native form for crystallization, thus a thrombin-cleavage site was inserted into WT CP3GT and 3 mutants to remove tags following purification. Some studies have suggested that the presence of tags alters enzyme activity, thus this presented the opportunity to test the effect of tags by assaying both native and tagged enzyme. Initial results showed that WT CP3GT treated with thrombin retained 70 percent activity after a 2-hour treatment at 4o C. Additional assays will be conducted to fully determine tag effects and will run concurrently with crystallization experiments