Arachidonoyl-carnitine and arachidonoyl-coenzyme A are suitable substrates for mammalian ALOX isoforms.

Abstract

Lipoxygenases (ALOX) convert free polyenoic fatty acids to bioactive mediators, which induce phenotypic alterations in target cells. However, the intracellular concentrations of free fatty acids are very low since these compounds are either rapidly esterified with coenzyme-A. The acyl-CoA esters are subsequently used for re-acylation via the Lands cycle or they are trans-esterified to acyl carnitines for mitochondrial import. Whether acyl carnitines and acyl-CoA derivatives might also serve as ALOX substrates has not been explored. In the present study, we prepared six different wildtype mammalian ALOX-isoforms and a selected enzyme mutant, incubated the recombinant proteins in vitro with free arachidonic acid, arachidonoyl-carnitine and arachidonoyl-coenzyme A and quantified the amounts of primary oxygenation products. We found that for most ALOX-isoforms arachidonoyl-carnitine was oxygenated with a similar rate as free arachidonic acid and that the chemical structures of the primary oxygenation products were identical. In contrast, arachidonoyl-coenzyme A was oxygenated with a 3-5-fold lower rate but here again highly specific patterns of primary oxygenation products were formed. In silico docking studies and molecular dynamics simulations suggested that free arachidonic acid and arachidonoyl-carnitine are similarly aligned at the active site of rabbit ALOX15 but binding of arachidonoyl-coenzyme A was sterically hindered because of the bulkiness of the CoA moiety. Taken together, our data indicate that acyl carnitines and fatty acid coenzyme A esters are suitable lipoxygenase substrates and that these compounds are oxygenated to isoform-specific patterns of primary oxygenation products.