Isolation and characterization of a cDNA encoding a Δ⁸ sphingolipid desaturase from Aquilegia vulgaris

We have isolated a cDNA encoding the Δ⁸ sphingolipid desaturase from the plant Aquilegia vulgaris L. via a PCR-based strategy using primers designed to target the conserved histidine box regions of microsomal desaturases. The function of the cDNA was confirmed by expression in the yeast, Saccharomyces cerevisiae. Analysis of the long-chain sphingoid bases as their dinitrophenyl derivatives by reverse-phase HPLC demonstrated the accumulation of cis- and trans-desaturated sphingoid bases which were not present in the wild-type yeast cells. The Δ⁸ desaturated products co-eluted with known Δ⁸-desaturated phytosphingenine and the molecular mass of these products was confirmed by liquid chromatography-MS. The Δ⁸ long-chain base desaturase was also able to desaturate dihydrosphingosine substrates. This is the first report of the functional characterization of an A. vulgaris gene product.