title: A cytochrome-b5-containing fusion protein similar to plant acyl lipid desaturases.
abstract: 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.
authors.affiliation: Institut für Allgemeine Botanik, Universität Hamburg, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Sperling
authors.forename: H
authors.initials: H
authors.lastname: Schmidt
authors.forename: E
authors.initials: E
authors.lastname: Heinz
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015786:Cytochromes b5 / Q000737:chemistry* / Q000378:metabolism; D017931:DNA Primers / Q000138:chemical synthesis; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000737:chemistry*; D006368:Helianthus / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D012639:Seeds / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D015786:Cytochromes b5; D044943:Fatty Acid Desaturases