Investigation of conserved cysteines on 6 fatty acid desaturase function in Arabidopsis thaliana

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Parth Dave (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Plants are an incredibly diverse group of organisms with over 300,000 species inhabiting different environments worldwide. Recent advances in biotechnology have driven investigations into the biochemical properties of plant systems to improve agricultural production in dynamic environments. The goal of my project is to test the effect of selected amino acid mutations on the FAD2 enzyme from Arabidopsis thaliana, a model system for plants and seed oil production. FAD2, a delta 12 fatty acid desaturase (FAD), is an essential plant enzyme that introduces a carbon-carbon double bond at the 12th position from the carboxyl end of an 18:1 fatty acid. We have identified seven different cysteine residues within the protein sequence of FAD2 that are well conserved in planta, suggesting that they may be essential to the functionality of the enzyme. Cysteine residues are often involved in catalysis and stabilizing the tertiary structure of proteins through disulfide bond formation. Each of these cysteine residues has been substituted for the amino acid alanine which cannot form disulfide bonds and/or render it incapable of carrying out catalysis or other structural roles. I hypothesized that each mutation would result in a FAD2 enzyme with reduced activity, likely due to decreased protein stability. The activity was tested by expressing the coding sequence for FAD2 for each of these mutants in yeast cells. Based on preliminary evidence, three out of seven designed mutants exhibited a significant decrease in (%) total lipid production levels. Ultimately, the FAD2 site-directed mutants will be tested in Arabidopsis mutant lines to ultimately gain additional insight on the structure-function of these enzymes, primarily focusing on plant oil production.

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

FAD2;cysteine;site-directed mutagenesis

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