A cytochrome-b5-containing fusion protein similar to plant acyl lipid desaturases

Eur J Biochem. 1995 Sep 15;232(3):798-805.


The similarity between oleate and linoleate desaturase sequences from several plants was used to construct degenerate oligonucleotide primers for PCR experiments with cDNA transcribed from mRNA of ripening sunflower embryos. A DNA fragment was amplified and sequenced. Specific primers derived from this partial sequence were used for rapid amplification of the 3'- and 5'-ends of this cDNA. With appropriate primers derived from these sequences, a full-length clone of 1377 bp was amplified by PCR which, after sequencing, showed an open reading frame of 458 amino acids corresponding to a putative protein of about 52 kDa. Comparison with other desaturases showed the conserved three histidine boxes and the characteristic hydropathy profile of membrane-bound desaturases, but the amino acid identity was restricted to 18% and the N-terminal region was about 100 amino acids longer. This N-terminal extension showed high similarity with cytochrome b5 and, accordingly, the whole sequence can be considered as coding for a fusion protein between cytochrome b5 and a desaturase-like enzyme. Furthermore, we detected a similar cytochrome b5 fold in the previously sequenced delta 9 acyl-CoA desaturase from yeast, but in this enzyme it was located at the C-terminus. An alignment of these fusion proteins with other heme-binding proteins revealed desaturases to be novel members of the cytochrome b5 superfamily. A truncated DNA representing 366 bp of the 5'-end was amplified from the cDNA clone and expressed in Escherichia coli. The truncated cDNA coded for a soluble protein of about 12 kDa as shown by SDS/PAGE and N-terminal sequencing. The enriched recombinant protein exhibited redox absorbance spectra characteristic of plant microsomal cytochrome b5.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Cytochromes b5 / chemistry*
  • Cytochromes b5 / metabolism
  • DNA Primers / chemical synthesis
  • DNA, Complementary
  • Escherichia coli / genetics
  • Fatty Acid Desaturases / chemistry*
  • Helianthus / enzymology*
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Recombinant Proteins / biosynthesis
  • Seeds / genetics
  • Sequence Homology, Amino Acid
  • Spectrophotometry


  • DNA Primers
  • DNA, Complementary
  • Recombinant Proteins
  • Cytochromes b5
  • Fatty Acid Desaturases

Associated data

  • GENBANK/X87143