BIOSYNTHESIS OF CONJUGATED TRIENE-CONTAINING FATTY-ACIDS BY A NOVEL ISOMERASE FROM THE RED MARINE ALGA PTILOTA-FILICINA

The biosynthesis of conjugated triene-containing fatty acids by the re d alga Ptilota filicina is catalyzed by a novel enzyme, polyenoic fatt y acid isomerase. The enzyme has been highly purified and is described here for the first time. Matrix-assisted laser-induced desorption mas s spectrometry was used to determine that the major protein in the pur ified enzyme is composed of similar or identical subunits of M(r) 58 1 19 Da. The native enzyme emerges with an apparent M(r) of 174 000 Da f rom a gel permeation chromatography column. While this enzyme catalyze s the formation of conjugated trienes from a variety of polyunsaturate d fatty acid precursors [arachidonate ((5Z,8Z,11Z,14Z)-eicosatetraenoa te) is converted to (5Z,7E,9E,14Z)-eicosatetraenoate; gamma-linolenate ((6Z,9Z,12Z)-octadecatrienoate) is converted to 6Z,8E,-10E-octadecatr ienoate], this occurs most rapidly with eicosapentaenoate [(5Z,7E,9E,1 4Z,17Z)-eicosapentaenoate], which is likely the native substrate. Thro ugh a series of experiments utilizing gamma-linolenates stereospecific ally labeled with deuterium, we have determined that the enzyme intram olecularly transfers the bis-allylic pro-S hydrogen from the C11 posit ion to the C13 position. Furthermore, the bis-allylic pro-R hydrogen a t C8 in gamma-linolenate is lost to the solvent. Using arachidonate as substrate, we demonstrated that the C11 olefinic position becomes pro tonated by a solvent-derived proton. There appears to be no requiremen t for molecular oxygen, and the transformation is catalyzed by this si ngle enzyme.