Multiple Conserved Cysteine Residues Are Required for Delta‐12 Fatty Acid Desaturase Function in Arabidopsis thaliana

Abstract

Plants produce two essential fatty acids (FAs) for human nutrition: linoleic acid (18:2Δ9,12, an omega‐3 FA) and alpha‐linoleic acid (18:3Δ9,12,15, an omega‐6 FA). Plant FA desaturase 2 (FAD2) is a microsomal enzyme that introduces the carbon‐carbon double bond at the delta‐12 position through an oxygenated intermediate. As the structure of FAD2 has yet to be experimentally determined, we are using integrated structural modeling, sequence analyses, and biochemical approaches to better understand this essential membrane‐bound enzyme. For this project we are studying the role of cysteines (Cys), using Arabidopsis thaliana FAD2 as a model, given their potential involvement in disulfide bonds, catalysis, cofactor binding, and/or metal coordination. We have identified seven conserved Cys residues in planta that we hypothesize to play distinct roles in the structure and function of FAD2, including one located in a highly conserved catalytic histidine motif HXXXH. To elucidate their roles, each individual Cys has been mutated to alanine. Transient expression assays of FAD2 in yeast, followed by lipid compositional analysis by gas chromatography has demonstrated that all Cys mutations resulted in reduced enzymatic activity with two appearing essential. Analysis of homodimer protein‐protein interactions in yeast has identified one Cys required for dimerization and has highlighted potentially interacting regions. To expand upon these results, we are currently testing for the presence of intramolecular and intermolecular disulfide bonds and assessing protein stability over time. These results have demonstrated that several Cys are required for FAD2 function providing a new model for understanding the contribution of these residues to 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.