Investigating the Role of Conserved Cysteine Residues in Arabidopsis thaliana Diacylglycerol Acyltransferase 1

Abstract

Triacylglycerols (TAGs) are biomolecules found in all organisms comprised of three fatty acid chains esterified to a glycerol backbone. In humans, TAGs are important energy storage reserves whose misregulation are associated with diseases such as obesity, diabetes, and heart disease. TAGs are also important for plant growth and development, in particular for seed lipid (or oil) reserves, as decreased TAG production is associated with poor quality seeds incapable of germination. As extractable chemical feedstocks, TAGs are a major component of biodiesel production, a green fuel source that is domestically produced and burns cleaner than typical petroleum-based fuels. Biochemically, the most common and rate-limiting final step for generating TAGs requires a diacylglycerol acyltransferase (DGAT) enzyme. This enzyme esterifies a fatty acyl-CoA to a diacylglycerol molecule. Despite its importance, the membrane-spanning topology and hydrophobicity of DGATs has prevented determination of its three-dimensional structure until recently. Plant DGAT enzymes contain cysteine-associated motifs (e.g. CXXXXC) that are reminiscent of redox-regulated switches and may be important for DGAT structure/activity (and therefore TAG accumulation). We have performed site-directed mutagenesis on several highly conserved cysteines and tested the associated lipid production levels in the model systems yeast, tobacco, and Arabidopsis thaliana. The lipid amounts and compositions produced from the mutated enzymes, along with protein amounts from western blots, were compared to the wild-type to determine how the cysteine mutations affect DGAT-catalyzed TAG production. This study will inform the strategic bioengineering of DGATs that could lead to improvements in biodiesel production, animal feed, and specialized dietary supplements produced by oleaginous plants or microorganisms.