RESUMEN
The substrate specificity and regioselectivity of the Brassica napus extraplastidial linoleate desaturase (FAD3) was investigated in vivo in a heterologous expression system. A strain of the yeast Saccharomyces cerevisiae producing the plant enzyme was constructed and cultured in media containing a variety of fatty acids. The products of desaturation of these potential substrates were determined by gas chromatographic and mass spectrometric analysis of the yeast cultures. The results indicate that the enzyme has: (a) omega-3, as opposed to Delta-15 or double-bond-related regioselectivity, (b) the ability to desaturate substrates in the 16 to 22 carbon range, (c) a preference for substrates with omega-6 double bonds, but the ability to desaturate substrates with omega-6 hydroxyl groups or omega-9 or omega-5 double bonds, and (d) a relative insensitivity to double bonds proximal to the carboxyl end of the substrate.
Asunto(s)
Brassica/enzimología , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Secuencia de Bases , Brassica/genética , Cartilla de ADN/genética , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Expresión Génica , Genes de Plantas , Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Transformación GenéticaRESUMEN
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.
Asunto(s)
Brassica/enzimología , Caenorhabditis elegans/enzimología , Ácido Graso Desaturasas/química , Ácido Graso Desaturasas/metabolismo , Animales , Sitios de Unión , Clonación Molecular , Cromatografía de Gases y Espectrometría de Masas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Especificidad por SustratoRESUMEN
Sequences of three Arabidopsis thaliana and two Brassica napus cDNAs encoding squalene monooxygenase homologues (Sqp1 and Sqp2) are reported. Southern analysis confirmed that these cDNAs are derived from small gene families in both species. Expression analysis indicates that Sqp1 genes in B. napus are strongly expressed in leaves but not roots or developing seeds. Comparison of cDNA and genomic sequences indicate that the 3' splice site of an intron in these genes has undergone junctional sliding. The evolutionary significance of this phenomenon is discussed.