D. Meesapyodsuk et al., Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases, BIOCH SOC T, 28, 2000, pp. 632-635
In order to define the substrate requirements, regiochemistry and cryptoreg
iochemistry of the omega -3 fatty acid desaturases involved in polyunsatura
ted 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 indicat
e that both the plant and animal desaturases act on unsaturated substrates
of 16-20 carbons with a preference for omega -6-unsaturated fatty acids. Th
e 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 exp
eriments. 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 observe
d for the plant or the nematode enzymes. These results indicate that omega
-3 desaturation is initiated by an energetically difficult C-H bond cleavag
e at the carbon closer to the carboxyl terminus. These results will be disc
ussed in the context of a general model relating the structure and function
of membrane-bound fatty acid desaturases featuring different regioselectiv
ities.