Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase (FAT-1) expressed in Saccharomyces cerevisiae.
To characterize the fatty acid desaturase produced by the fat-1 gene from the nematode Caenorhabditis elegans, the functional expression of this enzyme was effected in the yeast Saccharomyces cerevisiae. The GC-MS analysis of desaturated products derived from various fatty acids, including deuterium-labeled thia fatty acids supplied to growing cultures of transformed yeast, has defined the substrate requirements, regiochemistry, and cryptoregiochemistry of the enzyme. The desaturase acts on substrates of 16-20 carbons with a preference for omega-6 fatty acids, and its regioselectivity was confirmed to be that of an omega-3 desaturase. (omega-x refers to a double bond or desaturation between carbons x and x+1, counting from the methyl end of a fatty acid.) The primary deuterium kinetic isotope effects (KIEs) at C-15 and C-16 of a C18 fatty acid analogue were measured via competitive incubation experiments: While k(H)/k(D) at the omega-3 position was shown to be large (7.8 +/- 0.4), essentially no KIE at the omega-2 position was observed (k(H)/k(D) = 0.99 +/- 0.04). This result indicates that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. The results are discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring differing regioselectivities.
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National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9.
Reed
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Savile
C K
CK
Buist
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Ambrose
S J
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Covello
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D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology*; D003903:Deuterium / Q000737:chemistry; D044943:Fatty Acid Desaturases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D005231:Fatty Acids, Unsaturated / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000235:genetics
D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; D003903:Deuterium; D044943:Fatty Acid Desaturases; C078565:omega-3 fatty acid desaturase