Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal ω-3 fatty acid desaturases

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the omega -3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast, The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16-20 carbons with a preference for omega -6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of omega -3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C-18 fatty acid analogue were measured via competitive incubation experiments. Whereas k(H)/k(D) at the omega -3 position was shown to be large, essentially no kinetic isotope effect at the omega -2 position was observed for the plant or the nematode enzymes. These results indicate that omega -3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.