Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-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.
Meesapyodsuk
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NRC Plant Biotechnology Institute, 110 Gymnasium Road, Saskatoon, SK, Canada S7N 0W9.
Reed
D W
DW
Savile
C K
CK
Buist
P H
PH
Schäfer
U A
UA
Ambrose
S J
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Covello
P S
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D000818:Animals; D001665:Binding Sites; D001937:Brassica / Q000201:enzymology*; D017173:Caenorhabditis elegans / Q000201:enzymology*; D003001:Cloning, Molecular; D044943:Fatty Acid Desaturases / Q000737:chemistry* / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases; C078565:omega-3 fatty acid desaturase