Cryptoregiochemistry of a Brassica napus fatty acid desaturase (FAD3): a kinetic isotope effect study

Abstract

α-Linolenic acid ((Z,Z,Z)-octadeca-9,12,15-trienoic acid) is biosynthesized by a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. As part of ongoing research into the mechanism of these remarkable catalysts, we have examined the cryptoregiochemistry (site of initial oxidation) of extraplastidial ω − 3 desaturation as it occurs in the commercially important plant Brassica napus (oilseed rape or canola). The individual deuterium kinetic isotope effects associated with the C–H bond cleavages at C-15 and C-16 of a thiaoleoyl analogue were measured using a convenient in vivo yeast expression system. Competition experiments using appropriately deuterium-labelled 7-thia substrates revealed a large kinetic isotope effect (KIE) (kH/kD = 7.5 ± 0.4) for the C–H bond-breaking step at C-15 while the C–H bond cleavage at C-16 was found to be insensitive to deuterium substitution (kH/kD = 1.0 ± 0.14). These results point to C-15 as the site of initial oxidation in ω − 3 desaturation since the first chemical step in this type of reaction is rupture of a strong, unactivated C–H bond—an energetically difficult process which typically exhibits a large KIE.