Biochemical and Molecular Characterization of ACH2, an Acyl-CoA Thioesterase from Arabidopsis thaliana

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 (-SKL COOH ), 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 mol/min/mg protein. Additionally, ACH2-6His is insensitive to feedback inhibition by free CoASH levels as high as 100 M. 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.

Patatin-Related Phospholipase pPLAIIIβ-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis

“…Information on plant acyl-CoA thioesterases is limited. One Arabidopsis peroxisomal acyl-CoA thioesterase, ACH2, has been cloned and biochemically characterized, and the data suggest that this enzyme is unlikely to be involved in fatty acid oxidation, but its function is unclear (Tilton et al, 2004). A thioesterase activity was previously reported for mammalian iPLA 2 b, which is also a patatin domain-containing enzyme (Jenkins et al, 2006).…”

“…However, the physiological roles of acyl-CoA thioesterases remain unclear. One role suggested for peroxisomal acyl-CoA thioesterases is in fatty acid oxidation (Tilton et al, 2004). Information on plant acyl-CoA thioesterases is limited.…”

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses

“…The final step of JA biosynthesis is that jasmonyl-CoA releases free acid by jasmonyl-thioesterase. There is no report of cloning of jasmonyl-thioesterase in plant so far except that in Arabidopsis two peroxyisomal acyl-thioesterases, ACH1 and ACH2 have showed thioesterase activity of hydrolyzing both medium and long-chain fatty acylCoAs but not jasmonyl-CoA (Tilton et al, 2004).…”

Molecular Cloning and Characterization of Two Mouse Peroxisome Proliferator-activated Receptor α (PPARα)-regulated Peroxisomal Acyl-CoA Thioesterases

“…In addition, peroxisomes contain another structurally unrelated acyl-CoA thioesterase, called PTE-2. This enzyme was initially identified as an HIV-1 Nefbinding protein (21,22) and subsequently further characterized as an acyl-CoA thioesterase in mouse, human, yeast, and rat (19,20,23,38). Characterization of PTE-2 in mouse showed it to be the major acyl-CoA thioesterase in peroxisomes, hydrolyzing a broad range of acyl-CoA esters.…”