Further characterization of Delta(8)-sphingolipid desaturases from higher plants

A previously cloned cDNA from Helianthus annuus codes for a fusion protein composed of an N-terminal cytochrome b(5) and a C-terminal desaturase domai n. For a functional identification, this cDNA was expressed in Saccharomyce s cerevisiae and the structures of sphingolipid long-chain bases were analy sed. The expression of this sunflower enzyme resulted in the formation of n ew Delta (8)-trans/cis-phytosphingenine from C-18- and C-20-phytosphinganin e present in wild-type yeast cells. To elucidate the substrate specificity, the recently cloned Delta (8)-sphingolipid desaturases from Arabidopsis th aliana and Brassica napus were expressed in the yeast mutant sur2 Delta tha t lacked the sphinganine C-4-hydroxylase and was thus unable to form phytos phinganine. Long-chain base analysis of the transformed mutant cells did no t show any conversion of C-18- or C-20-sphinganine into Delta (8)-sphingeni ne, whereas exogenously added C-18-phytosphinganine was desaturated to Delt a (8)-trans/cis-phytosphingenine. Furthermore, GLC-MS analysis did not reve al the presence of any Delta (9)-regioisomers as reported before. These res ults show that the sunflower gene codes for a Delta (8)-sphingolipid desatu rase which accepts C-18- and C-20-phytosphinganine. The absence of Delta (8 )-sphingenine as desaturation product in the transformed mutant suggests th at C-4-hydroxylation of sphinganine precedes Delta (8)-desaturation. Theref ore, in yeast, the substrate for the plant Delta (8)-sphingolipid desaturas e seems to be the phytosphinganine residue.