Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Delta4-fatty acyl group desaturase.
Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the omega3/omega6-pathways involve the action of a Delta4-fatty acid desaturase. We examined the suitability of Euglena gracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources for cloning a cDNA encoding a Delta4-fatty acid desaturase. For this purpose we carried out in vivo labeling studies with radiolabeled C22 polyunsaturated fatty acid substrates. Schizochytrium sp. was unable to convert exogenously supplied [2-(14)C]-docosapentaenoic acid (DPA, 22:5(Delta)(7,10,13,16,19)) to DHA, while E. gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and alpha-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Delta4-desaturation and not of substrate breakdown and resynthesis. To clone the desaturase gene, a cDNA library of E. gracilis was subjected to mass sequencing. A full-length clone with highest homology to the Delta4-desaturase of Thraustochytrium sp. was isolated, and its function was verified by heterologous expression in yeast. The desaturase efficiently converted DPA to DHA. Analysis of the substrate specificity demonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturated C16 fatty acids. The enzyme showed strict Delta4-regioselectivity and required the presence of a Delta7-double bond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the Delta4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.
Meyer
Astrid
A
Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany.
Cirpus
Petra
P
Ott
Claudia
C
Schlecker
Rainer
R
Zähringer
Ulrich
U
Heinz
Ernst
E
D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D004281:Docosahexaenoic Acids / Q000378:metabolism*; D005056:Euglena gracilis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000032:analysis; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism; D005658:Fungi / Q000235:genetics / Q000378:metabolism; D006865:Hydrogenation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity
D002247:Carbon Isotopes; D018076:DNA, Complementary; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D004281:Docosahexaenoic Acids; D044943:Fatty Acid Desaturases; C026219:docosapentaenoic acid