Further characterization of Δ8-sphingolipid desaturases from higher plants

Abstract

A previously cloned cDNA from Helianthus annuus codes for a fusion protein composed of an N-terminal cytochrome b5, and a C-terminal desaturase domain. For a functional identification, this cDNA was expressed in Saccharomyces cerevisiae and the structures of sphingolipid longchain bases were analysed. The expression of this sunflower enzyme resulted in the formation of new Δ8-trans/cis-phytosphingenine from C18- and C20-phytosphinganine present in wild-type yeast cells. To elucidate the substrate specificity, the recently cloned Δ8-sphingolipid desaturases from Arabidopsis thaliana and Brassica napus were expressed in the yeast mutant sur2Δ that lacked the sphinganine C4-hydroxylase and was thus unable to form phytosphinganine. Long-chain base analysis of the transformed mutant cells did not show any conversion of C18- or C20-sphinganine into Δ8-sphingenine, whereas exogenously added C18-phytosphinganine was desaturated to Δ8-trans/cis-phytosphingenine. Furthermore, GLC-MS analysis did not reveal the presence of any Δ9-regioisomers as reported before. These results show that the sunflower gene codes for a Δ8-sphingolipid desaturase which accepts C18- and C20-phytosphinganine. The absence of Δ8-sphingenine as desaturation product in the transformed mutant suggests that C4-hydroxylation of sphinganine precedes Δ8-desaturation. Therefore, in yeast, the substrate for the plant Δ8-sphingolipid desaturase seems to be the phytosphinganine residue.