|Tilton, Gregory - MICHIGAN STATE UNIV.|
|Browse, John - WASHINGTON STATE UNIV.|
Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 11, 2003
Publication Date: December 2, 2003
Citation: Tilton, G.B., Shockey, J.M., Browse, J. 2003. Biochemical and molecular characterization of ach2, an acyl-coa thioesterase from arabidopsis thaliana. Journal of Biological Chemistry. 279:(9)7487-7494. Interpretive Summary: Many of the chemical and physical properties of the membranes of all living cells, including plant cells, are determined by the fatty acid composition of membrane and storage lipids. Fatty acid composition is in part determined by the relative amounts of the activated fatty acid precursor molecules called acyl-CoAs. While various types of enzymes can affect the concentrations and composition of the cellular acyl-CoA pool, the class of enzymes known as acyl-CoA hydrolases (ACHs) may be one of the most important. ACH enzymes actually deacitivate fatty acids by removing the CoA group from a molecule of acyl-CoA. For this reason, scientists at Washington State University and the SRRC sought to study ACHs at the molecular level to learn more about their effects on acyl-CoA concentrations in plant cells. This paper describes the cloning and characterization of the first ACH gene from plants. The gene is called ACH2, and was isolated from the laboratory model plant Arabidopsis thaliana. The data presented in this paper represent the first glimpses into how ACHs fit into the overall scheme of lipid synthesis and breakdown. As such, these findings will broadly benefit all areas of plant lipid research, especially those researchers seeking to engineer traditional oilseed crops to produce new industrial and edible vegetable oils.
Technical Abstract: By using computer-based homology searches of the Arabidopsis genome, we identified the gene for ACH2, a putative acyl-CoA thioesterase. With the exception of a unique 129-amino acid N-terminal extension, the ACH2 protein is 17'36% identical to members of a family of acyl-CoA thioesterases that are found in both prokaryotes and eukaryotes. The eukaryotic homologs of ACH2 are peroxisomal acyl-CoA thioesterases that are up-regulated during times of increased fatty acid oxidation, suggesting potential roles in peroxisomal-oxidation. We investigated ACH2 to determine whether it has a similar role in the plant cell. Like its eukaryotic homologs, ACH2 carries a putative type 1 peroxisomal targeting sequence (-SKLCOOH), and maintains all the catalytic residues typical of this family of acyl-CoA thioesterases. Analytical ultracentrifugation of recombinant ACH2'6His shows that it associates as a 196-kDa homotetramer in vitro, a result that is significant in light of the cooperative kinetics demonstrated by ACH2'6His in vitro. The cooperative effects are most pronounced with medium chain acyl-CoAs, where the Hill coefficient is 3.8 for lauroyl-CoA, but decrease for long chain acyl-CoAs, where the Hill coefficient is only 1.9 for oleoyl-CoA. ACH2'6His hydrolyzes both medium and long chain fatty acyl-CoAs but has highest activity toward the long chain unsaturated fatty acyl-CoAs. Maximum rates were found with palmitoleoyl-CoA, which is hydrolyzed at 21 µmol/min/mg protein. Additionally, ACH2'6His is insensitive to feedback inhibition by free CoASH levels as high as 100 µM. ACH2 is most highly expressed in mature tissues such as young leaves and flowers rather than in germinating seedlings where -oxidation is rapidly proceeding. Taken together, these results suggest that ACH2 activity is not linked to fatty acid oxidation as has been suggested for its eukaryotic homologs, but rather has a unique role in the plant cell.