Investigating the Structural and Functional Roles of Conserved Cysteine Residues in Arabidopsis thaliana Fatty-Acid Desaturase 2

Abstract

Plant-generated unsaturated fatty acids (UFAs) and polyunsaturated fatty acids (PUFAs) are vital to a plant's growth and survival through maintenance of plasma membrane fluidity and integrity, in addition to the various functions lipids carry out within all organisms. These compounds are also in high demand due to human reliance on their incorporation into everyday diets, products, and the increasing field of research into biofuels. Fatty acid desaturases (FADs) are key enzymes in the synthesis of these biomolecules, but limited research into the diverse subgroup of membrane-bound desaturases has been performed. Plant fatty acid desaturase 2 (FAD2) is a microsomal enzyme that introduces a carbon-carbon double bond at the [delta]12 position of oleic acid through an oxygenated intermediate to form linoleic acid, and as a membrane-bound desaturase it remains structurally uncharacterized. Sequence comparison of FAD2 from model organism Arabidopsis thaliana to plant species expressing homologous proteins has uncovered seven cysteines conserved to various degrees that we hypothesized to play distinct roles in the structure and function of FAD2. To evaluate this concept, site-directed mutagenesis of each cysteine to alanine was performed followed by transient expression of FAD2 in Saccharomyces cerevisiae. Preliminary lipid compositional analysis by gas chromatography has demonstrated that each cysteine mutation resulted in reduced enzymatic activity with two mutations completely depleting activity. These results brought into question the involvement of each cysteine in the established homodimer protein-protein interaction and overall protein stability. By determining the roles of the conserved cysteines within FAD2, more will be understood about the enzyme structure and mechanism which can be applied to other similarly uncharacterized membrane-bound FADs in plant systems. Ultimately, a better understanding of these enzymes will inform rational engineering strategies for producing essential lipids and high value bioproducts.