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

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