Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases.
نویسندگان
چکیده
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.
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ورودعنوان ژورنال:
- Biochemical Society transactions
دوره 28 6 شماره
صفحات -
تاریخ انتشار 2000