title: Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases.
abstract: 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.
authors.affiliation: NRC Plant Biotechnology Institute, 110 Gymnasium Road, Saskatoon, SK, Canada S7N 0W9.
authors.forename: D
authors.initials: D
authors.lastname: Meesapyodsuk
authors.forename: D W
authors.initials: DW
authors.lastname: Reed
authors.forename: C K
authors.initials: CK
authors.lastname: Savile
authors.forename: P H
authors.initials: PH
authors.lastname: Buist
authors.forename: U A
authors.initials: UA
authors.lastname: Schäfer
authors.forename: S J
authors.initials: SJ
authors.lastname: Ambrose
authors.forename: P S
authors.initials: PS
authors.lastname: Covello
mesh_terms: 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
chemical_list: D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases; C078565:omega-3 fatty acid desaturase