tailieunhanh - Báo cáo Y học: Mechanism of 1,4-dehydrogenation catalyzed by a fatty acid (1,4)-desaturase of Calendula officinalis

The mechanism by which the fatty acid (1,4)-desaturase of Calendulaofficinalisproduces calendicacid fromlinoleicacid has been probed through the use of kinetic isotope effect (KIE) measurements. This was accomplished by incubating appropriate mixtures of linoleate and regiospecifically dideuterated isotopomers with a strain ofSaccharomyces cerevisiaeexpressing a functional (1,4)-desaturase. | Eur. J. Biochem. 269 5024-5029 2002 FEBS 2002 doi Mechanism of 1 4-dehydrogenation catalyzed by a fatty acid 1 4 -desaturase of Calendula officinalis Darwin W. Reed1 Christopher K. Savile2 Xiao Qiu1 Peter H. Buist2 and Patrick S. Covello1 1 Plant Biotechnology Institute Saskatoon SK Canada department of Chemistry Carleton University Ottawa Ontario Canada The mechanism by which the fatty acid 1 4 -desaturase of Calendula officinalis produces calendic acid from linoleic acid has been probed through the use of kinetic isotope effect KIE measurements. This was accomplished by incubating appropriate mixtures of linoleate and regiospecifically dideuterated isotopomers with a strain of Saccharomyces cerevisiae expressing a functional 1 4 -desaturase. GC-MS analysis of methyl calendate obtained in these experiments showed that the oxidation of linoleate occurs in two discrete steps since the cleavage of the C11-H bond is very sensitive to isotopic substitution kH kD while no isotope effect kH kD was observed for the C8-H bond breaking step. These data indicate that calendic acid is produced via initial H-atom abstraction at C11 of a linoleoyl substrate and supports the hypothesis that this transformation represents a regiochemical variation of the more common C12-initiated A12 desaturation process. Keywords desaturase kinetic isotope effect conjugated fatty acid deuterium labelling Calendula. The A12-oleate desaturase FAD2 family of enzymes are membrane-bound nonheme iron-containing proteins that carry out a fascinating array of related oxidative transformations 1 2 . The prototypical reaction features the introduction of a cis-double bond at the 12 13-position of an oleoyl substrate - a ubiquitous biosynthetic reaction of higher plants 3 4 Fig. 1A . Species-specific mechanistic variations of this process include 12-hydroxylation of oleate Ricinus communis 5 and 12 13-epoxidation Crepis palaestina 2 or 12 13-acetylenation .