Functional identification of a delta8-sphingolipid desaturase from Borago officinalis.
The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves.
Sperling
P
P
Institut für Allgemeine Botanik, Universität Hamburg, Germany.
Libisch
B
B
Zähringer
U
U
Napier
J A
JA
Heinz
E
E
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019684:Magnoliopsida / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000096:biosynthesis
D010088:Oxidoreductases; C115532:sphingolipid desaturase; D013110:Sphingosine; C005682:safingol