Meesapyodsuk D et al. (2000) Biochemistry "Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase (FAT-1) expressed in Saccharomyces cerevisiae."

Buist PH, Savile CK, Ambrose SJ, Meesapyodsuk D, Reed DW, Covello PS

[Biochemistry, 2000]

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.

Meesapyodsuk D et al. (2000) Biochem Soc Trans "Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases."

Buist PH, Covello PS, Reed DW, Ambrose SJ, Schafer UA, Savile CK, Meesapyodsuk D

[Biochem Soc Trans, 2000]

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.

aldose beta-D-fructosyltransferase activity

Catalysis of the reaction: alpha-D-aldosyl1 beta-D-fructoside + D-aldose2 = D-aldose1 + alpha-D-aldosyl2 beta-D-fructoside.

lacto-N-biosidase activity

Catalysis of the reaction: H2O + beta-D-Gal-(1,3)-beta-D-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc = beta-D-Gal-(1,4)-D-Glc + beta-D-Gal-(1,3)-D-GlcNAc.

mannotetraose 2-alpha-N-acetylglucosaminyltransferase activity

Catalysis of the reaction: 1,3-alpha-D-mannosyl-1,2-alpha-D-mannosyl-1,2-alpha-D-mannosyl-D-mannose + UDP-N-acetyl-D-glucosamine = 1,3-alpha-D-mannosyl-1,2-(N-acetyl-alpha-D-glucosaminyl-alpha-D-mannosyl)-1,2-alpha-D-mannosyl-D-mannose + UDP.

globoside alpha-N-acetylgalactosaminyltransferase activity

Catalysis of the reaction: N-acetyl-D-galactosaminyl-(1,3)-D-galactosyl-(1,4)-D-galactosyl-(1,4)-D-glucosylceramide + UDP-N-acetylgalactosamine = N-acetyl-D-galactosaminyl-N-acetyl-D-galactosaminyl-(1,3)-D-galactosyl-(1,4)-D-galactosyl-(1,4)-D-glucosylceramide + UDP.

dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase activity

Catalysis of the reaction: an alpha-D-man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-(alpha-D-Man-(1->6))-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc-diphosphodolichol + dolichyl D-mannosyl phosphate = H+ + alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-(alpha-D-Man-(1->3)-alpha-D-Man-(1->6))-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc-diphosphodolichol + dolichyl phosphate.

glucosaminylgalactosylglucosylceramide beta-galactosyltransferase activity

Catalysis of the reaction: N-acetyl-D-glucosaminyl-(1,3)-D-galactosyl-(1,4)-D-glucosylceramide + UDP-galactose = D-galactosyl-N-acetyl-D-glucosaminyl-(1,3)-D-galactosyl-(1,4)-D-glucosylceramide + UDP.

glucose-fructose oxidoreductase activity

Catalysis of the reaction: D-fructose + D-glucose = D-glucitol + D-glucono-1,5-lactone.

dol-P-Man:Man(6)GlcNAc(2)-PP-Dol alpha-1,2-mannosyltransferase activity

Catalysis of the reaction: alpha-D-man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-(alpha-D-Man-(1->3)-alpha-D-Man-(1->6))-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc-diphosphodolichol + dolichyl D-mannosyl phosphate = H+ + alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-(alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-alpha-D-Man-(1->6))-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc-diphosphodolichol + dolichyl phosphate.