The Delta8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids.
Desaturation of fatty acids is an important metabolic process. In mammals, 20-carbon and longer polyunsaturated fatty acids are not only incorporated into cellular membranes in a tissue-specific manner, but also serve as the precursors to synthesis of eicosanoid metabolic regulators. The processes of desaturation and elongation in human liver are well characterized, but an alternate Delta8 desaturation pathway that may be important in certain tissues or in cancer cells is less well examined. The Delta8-desaturase enzyme introduces a double bond at the 8-position in 20-carbon fatty acids that have an existing Delta11 unsaturation. We have isolated the first fatty acid Delta8-desaturase, from the protist Euglena gracilis, in order to explore this alternate pathway. A full-length cDNA was obtained after reverse transcription of mRNA purified from heterotrophically grown Euglena, followed by PCR amplification with primers degenerate to conserved histidine-rich regions of microsomal desaturases. The protein predicted from the cDNA sequence is highly homologous to Delta5 and Delta6 desaturases of Caenhorabditis elegans. When the cDNA was expressed in Saccharomyces cerevisiae, the yeast cultures readily desaturated appropriate 20-carbon fatty acids by inserting an additional double bond at the Delta8-position. The enzyme demonstrated a preference for substrates of metabolic significance, 20:3 Delta11,14,17 and 20:2 Delta11,14. Cloning of a Delta8 fatty acid desaturase offers the opportunity to examine an alternate pathway of long chain fatty acid biosynthesis.
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005056:Euglena gracilis / Q000201:enzymology*; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis*; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity