A CYTOCHROME-B(5)-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 primer s for PCR experiments with cDNA transcribed from mRNA of ripening sunf lower embryos. A DNA fragment was amplified and sequenced. Specific pr imers derived from this partial sequence were used for rapid amplifica tion of the 3'- and 5'-ends of this cDNA. With appropriate primers der ived from these sequences, a full-length clone of 1377 bp was amplifie d by PCR which, after sequencing, showed an open reading frame of 458 amino acids corresponding to a putative protein of about 52 kDa.Compar ison with other desaturases showed the conserved three histidine bares and the characteristic hydropathy profile of membrane-bound desaturas es, but the amino acid identity was restricted to 18% and the N-termin al region was about 100 amino acids longer. This N-terminal extension showed high similarity with cytochrome b(5) and, accordingly, the whol e sequence can be considered as coding for a fusion protein between cy tochrome b(5) and a desaturase-like enzyme. Furthermore, we detected a similar cytochrome b(5) fold in the previously sequenced Delta(9) acy l-CoA desaturase from yeast, but in this enzyme it was located at the C-terminus. An alignment of these fusion proteins with other heme-bind ing proteins revealed desaturases to be novel members of the cytochrom e b(5) superfamily. A truncated DNA representing 366 bp of the 5'-end was amplified from the cDNA clone and expressed in Escherichia coli. T he truncated cDNA coded for a soluble protein of about 12 kDa as shown by SDS/PAGE and N-terminal sequencing. The enriched recombinant prote in exhibited redox absorbance spectra characteristic of plant microsom al cytochrome b(5).