A cytochrome-b5-containing fusion protein similar to plant acyl lipid desaturases.
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.
Sperling
P
P
Institut für Allgemeine Botanik, Universität Hamburg, Germany.
Schmidt
H
H
Heinz
E
E
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
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D015786:Cytochromes b5; D044943:Fatty Acid Desaturases