Molecular cloning and biochemical characterization of acyl-coenzyme A thioesterases from Arabidopsis thaliana

Acyl-coenzyme A thioesterases hydrolyze acyl-coenzyme A molecules to free fatty acids and free coenzyme A (CoA). Five different families of acyl-CoA thioesterases have been cloned and characterized from prokaryotic and eukaryotic organisms. The potential to regulate intracellular levels of acyl-CoAs makes acyl-CoA thioesterases an interesting subject of study, especially since acyl-CoA molecules are important intermediates in pathways such as fatty acid biosynthesis, lipid construction, and fatty acid degradation. A relationship between fatty acid degradation and acyl-CoA thioesterase activity has been established in yeast and mammals, where increased β-oxidation leads to upregulation of acyl-CoA thioesterase genes.; While acyl-CoA thioesterases have been characterized from prokaryotes and animals, very little is known about their role in plant lipid metabolism. In order to define the part that acyl-CoA thioesterases play in the plant cell, we conducted a search of the Arabidopsis thaliana genome in order to identify putative acyl-CoA thioesterase genes. Four different genes were discovered by the homology of their putative gene products with other known acyl-CoA thioesterases. We named these genes ACH1, ACH2, ACH4, and ACH5.; In order to study the role of the ACH proteins in the plant cell, we characterized the acyl-CoA thioesterase activity of recombinant ACH proteins. This characterization showed that ACH2 and ACH5 prefer acyl-CoA substrates longer than ten carbons, but ACH4 is capable of hydrolyzing short and long-chain acyl-CoAs.; In addition to biochemical characterization, T-DNA insertional mutants for each of the ACH genes were identified and analyzed. Growth studies of single and double mutants under a variety of conditions show that loss of the acyl-CoA thioesterase activity has no observable effect on the development of the plant compared to wild-type Arabidopsis. The lack of phenotype suggests that plant acyl-CoA thioesterases are not necessary for efficient β-oxidation or lipid biosynthesis. Instead, our results indicate that plant acyl-CoA thioesterases most likely have specific roles in the cell that are necessary under a specific set of circumstances. Discovery of additional acyl-CoA thioesterases and continued characterization of the mutants will be useful in revealing how plant acyl-CoA thioesterases are involved in the metabolism of fatty acids.