Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma.
The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.
Hornung
Ellen
E
Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, D-06466 Gatersleben, Germany.
Korfei
Martina
M
Pernstich
Christian
C
Struss
Annett
A
Kindl
Helmut
H
Fulda
Martin
M
Feussner
Ivo
I
D016718:Arachidonic Acid / Q000032:analysis / Q000096:biosynthesis*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D000090943:Delta-5 Fatty Acid Desaturase; D015118:Eicosapentaenoic Acid / Q000032:analysis / Q000096:biosynthesis*; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000032:analysis / Q000096:biosynthesis; D010838:Phytophthora / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000502:physiology; D013379:Substrate Specificity
D018076:DNA, Complementary; D000090943:Delta-5 Fatty Acid Desaturase; D005231:Fatty Acids, Unsaturated; D016718:Arachidonic Acid; D015118:Eicosapentaenoic Acid; D044943:Fatty Acid Desaturases