Open AccessMechanism of 1,4‐dehydrogenation catalyzed by a fatty acid (1,4)‐desaturase of Calendula officinalis
Author(s) -
Reed Darwin W,
Savile Christopher K.,
Qiu Xiao,
Buist Peter H.,
Covello Patrick S.
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.03209.x
Subject(s) - isotopomers , linoleic acid , calendula officinalis , chemistry , kinetic isotope effect , stereochemistry , double bond , ozonolysis , fatty acid , dehydrogenation , catalysis , biochemistry , organic chemistry , biology , botany , physics , deuterium , quantum mechanics , molecule
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 ( k H / k D = 5.7 ± 1.0) while no isotope effect ( k H / k D = 1.0 ± 0.1) 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 Δ 12 desaturation process.