Isolation and characterization of a cDNA encoding a Delta8 sphingolipid desaturase from Aquilegia vulgaris.
We have isolated a cDNA encoding the Delta(8) 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 Delta(8) desaturated products co-eluted with known Delta(8)-desaturated phytosphingenine and the molecular mass of these products was confirmed by liquid chromatography-MS. The Delta(8) 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.
Michaelson
L V
LV
Institute of Arable Crops Research, Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, UK. louise.michaelson@bbsrc.ac.uk
Longman
A J
AJ
Sayanova
O
O
Stobart
A K
AK
Napier
J A
JA
D031884:Aquilegia / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D018076:DNA, Complementary / Q000378:metabolism*; D015723:Gene Library; D008861:Microsomes / Q000201:enzymology; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D003062:Codon; D018076:DNA, Complementary; D010088:Oxidoreductases; C115532:sphingolipid desaturase