A bifunctional Δ6-fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus -: A new member of the cytochrome b5 superfamily

Many plant genes have been cloned that encode regioselective desaturases catalyzing the formation of cis-unsaturated fatty acids. However, very few genes have been cloned that encode enzymes catalyzing the formation of the functional groups found in unusual fatty acids (e.g. hydroxy, epoxy or acetylenic fatty acids). Here, we describe the characterization of an acetylenase from the moss Ceratodon purpureus with a regioselectivity differing from the previously described Delta(12)-acetylenase. The gene encoding this protein, together with a Delta(6)-desaturase, was cloned by a PCR-based approach with primers derived from conserved regions in Delta(5) -, Delta(6)-fatty-acid desaturases and Delta(8)-sphingolipid desaturases. The proteins that are encoded by the two cloned cDNAs are likely to consist of a N-terminal extension of unknown function, a cytochrome b(5)-domain, and a C-terminal domain that is similar to acyl lipid desaturases with characteristic histidine boxes. The proteins were highly homologous in sequence to the Delta(6)-desaturase from the moss Physcomitrella patens. When these two cDNAs were expressed in Saccharomyces cerevisiae, both transgenic yeast cultures desaturated Delta(9)-unsaturated C16- and C18-fatty acids by inserting an additional Delta(6)-cis-double bond. One of these transgenic yeast clones was also able to introduce a Delta(6)-triple bond into gamma-linolenic and stearidonic acid. This resulted in the formation of 9,12,15-(Z,Z,Z)-octadecatrien-6-ynoic acid, the main fatty acid found in C. pupureus. These results demonstrate that the Delta(6)-acetylenase from C. pupureus is a bifunctional enzyme, which can introduce a Delta(6)-cis-double bond into 9,12,(15)-C18-polyenoic acids as well as converting a Delta(6)-cis-double bond to a Delta(6)-triple bond.