Intermediates and products formed during fatty acid alpha-oxidation in cucumber (Cucumis sativus)

Fatty acid alpha-oxidation is an essential metabolic pathway both in plants and in mammals which is still not completely understood. We previously described and purified an alpha-oxidation enzyme in cucumber which has been used in the present investigation of the alpha-oxidation reaction mechanism. Free fatty acids, and not the CoA thioesters, were found to undergo alpha-oxidation in cucumber. 2-Hydroxy- and 2-oxopalmitic acids were identified as palmitic acid alpha-oxidation intermediates by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis in cucumber subcellular 150,000 x g(max) pellets obtained by differential centrifugation. Incubation of purified alpha-oxidation enzyme with [1-14C]palmitic acid resulted in the formation of both the above-described intermediates and the Cn-1 product, pentadecanal, and 14CO2. Besides 14CO2, 14C-formate was identified as an alpha-oxidation product from [1-14C]palmitic acid in cucumber subcellular fractions. Fe2+ stimulated the 14CO2 and 14C-formate production, and the addition of ascorbate and 2-oxoglutarate together with Fe2+ resulted in optimal alpha-oxidation activities, suggesting a dioxygenase reaction mechanism, as previously shown in mammals. NADPH and, to a lesser extent, NADH stimulated the total 14C-formate plus 14CO2 production but had only slight or no effects on 14CO2 production. H2O2 showed concentration-dependent inhibitory effects, while FAD had neither effect on 14CO2 nor 14CO2 plus 14C-formate production. The results in the present study demonstrate that an alpha-oxidation enzyme in cucumber is capable of oxidizing palmitic acid via 2-hydroxy- and 2-oxo-palmitic acid to produce pentadecanal and CO2. In contrast to the subcellular 150,000 x g(max) fraction, the purified alpha-oxidation enzyme could neither produce formate nor convert 14C-formate into 14C02, indicating two possible alpha-oxidation routes in cucumber.