Functional identification of a Δ8-sphingolipid desaturase from Borago officinalis

Petra Sperling, Ernst Heinz, Balázs Libisch, Johnathan A. Napier, Ulrich Zähringer

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

The similarities between Δ12- and Δ15-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 Δ8-sphingolipid desaturases as well as to the Δ6-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 Δ8-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-0-trimethylsilyl ether derivatives confirmed the presence of Δ8-stereo-isomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective Δ8-sphingolipid desaturase is primarily expressed in young borage leaves.

Original languageEnglish
Pages (from-to)293-298
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume388
Issue number2
DOIs
Publication statusPublished - Apr 15 2001

Keywords

  • Borago officinalis
  • Cytochrome b
  • Desaturase
  • GLC-MS
  • Phytosphingenine
  • Reversed-phase HPLC
  • Saccharomyces cerevisiae
  • Sphingolipids

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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