The Δ8-Desaturase ofEuglena gracilis:An Alternate Pathway for Synthesis of 20-Carbon Polyunsaturated Fatty Acids

Abstract

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 Δ8desaturation pathway that may be important in certain tissues or in cancer cells is less well examined. The Δ8-desaturase enzyme introduces a double bond at the 8-position in 20-carbon fatty acids that have an existing Δ11 unsaturation. We have isolated the first fatty acid Δ8-desaturase, from the protistEuglena gracilis,in order to explore this alternate pathway. A full-length cDNA was obtained after reverse transcription of mRNA purified from heterotrophically grownEuglena,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 Δ5and Δ6desaturases ofCaenhorabditis elegans.When the cDNA was expressed inSaccharomyces cerevisiae,the yeast cultures readily desaturated appropriate 20-carbon fatty acids by inserting an additional double bond at the Δ8-position. The enzyme demonstrated a preference for substrates of metabolic significance, 20:3 Δ11,14,17 and 20:2 Δ11,14. Cloning of a Δ8fatty acid desaturase offers the opportunity to examine an alternate pathway of long chain fatty acid biosynthesis.