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 domain. For a functional identification, this cDNA was expressed in Saccharomyces cerevisiae and the structures of sphingolipid long-chain bases were analysed. The expression of this sunflower enzyme resulted in the formation of new Delta(8)-trans/cis-phytosphingenine from C(18)- and C(20)-phytosphinganine present in wild-type yeast cells. To elucidate the substrate specificity, the recently cloned Delta(8)-sphingolipid desaturases from Arabidopsis thaliana and Brassica napus were expressed in the yeast mutant sur2Delta that lacked the sphinganine C(4)-hydroxylase and was thus unable to form phytosphinganine. Long-chain base analysis of the transformed mutant cells did not show any conversion of C(18)- or C(20)-sphinganine into Delta(8)-sphingenine, whereas exogenously added C(18)-phytosphinganine was desaturated to Delta(8)-trans/cis-phytosphingenine. Furthermore, GLC-MS analysis did not reveal the presence of any Delta(9)-regioisomers as reported before. These results show that the sunflower gene codes for a Delta(8)-sphingolipid desaturase which accepts C(18)- and C(20)-phytosphinganine. The absence of Delta(8)-sphingenine as desaturation product in the transformed mutant suggests that C(4)-hydroxylation of sphinganine precedes Delta(8)-desaturation. Therefore, in yeast, the substrate for the plant Delta(8)-sphingolipid desaturase seems to be the phytosphinganine residue.
Sperling
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P
Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststr. 18, 22609 Hamburg, Germany.
Blume
A
A
Zähringer
U
U
Heinz
E
E
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism; D006368:Helianthus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015786:Cytochromes b5; D010088:Oxidoreductases; C115532:sphingolipid desaturase