Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids

Abstract

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of ␥-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two ⌬6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina ⌬5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

Zebrafish cDNA Encoding Multifunctional Fatty Acid Elongase Involved in Production of Eicosapentaenoic (20:5n-3) and Docosahexaenoic (22:6n-3) Acids

“…Functional characterisation has previously been reported for PUFA elongases of nematode (C. elegans), fungus, (M. alpina), rat and human with all four enzymes being predominantly active on C 18 PUFA (Beaudoin et al, 2000;Leonard et al, 2000b;Parker-Barnes et al, 2000;Inagaki et al 2002). This was also the case with zebrafish elongase, the enzyme achieving 90% conversion of 18:4n-3 substrate and 60% of 18:3n-6.…”

“…The zebrafish enzyme also had substantial C 20 PUFA elongase activity, converting some 46% and 26% of 20:5n-3 and 20:4n-6, respectively, to the respective C 22 products. This is similar to human (ELOVL5) and rat elongases (rELO1), which also have high activity on 20:5n-3 and 20:4n-6 (Leonard et al, 2000b: Inagaki et al, 2002, but in contrast to the elongases of M. alpina and C. elegans which show virtually no activity towards C 20 PUFA (Parker-Barnes et al, 2000;Beaudoin et al, 2000). However, in contrast to the previously reported human and rat elongases described above, the zebrafish elongase also displayed the capacity to elongate C 22 PUFA, converting about 5% of 22:5n-3 to 24:5n-3 in the recombinant yeast system studied.…”

“…Biochemical studies have suggested that different elongase enzymes are involved in the elongation of saturated and unsaturated fatty acids (Sprecher, 1974;Prasad et al, 1986) and that there may be different enzymes catalysing the elongation of C 18/20 PUFA and C 22 PUFA (Luthria and Sprecher, 1997). Recently, enzymes catalyzing the elongation of C 18 PUFA have been cloned from the fungus Mortierella alpina (Parker-Barnes et al, 2000), the nematode Caenorhabditis elegans (Beaudoin et al, 2000), and humans (Leonard et al, 2000b). Variation in HUFA biosynthesis may also operate at the elongation steps as supported by the low C 18-20 elongase activity in the fish species turbot (Scophthalmus maximus) (Ghioni et al, 1999), a carnivorous marine teleost that requires a dietary supply of HUFA for normal growth (Bell et al, 1985).…”

Specific and differential inhibition of very-long-chain fatty acid elongases from Arabidopsis thaliana by different herbicides

“…Notably, fatty acid elongase (FAE)-like elongases are unique to plants (6). In animals and fungi, ELO-type elongases synthesize the fatty acids specific to sphingolipid synthesis (22)(23)(24)(25). All 21 Arabidopsis elongase genes were amplified by PCR using leaf and inflorescence cDNA as a template.…”

Effects of diets containing vegetable oil on expression of genes involved in highly unsaturated fatty acid biosynthesis in liver of Atlantic salmon (Salmo salar)

“…A fatty acid desaturase has been cloned from zebrafish that has been shown to have both Δ6 and Δ5 desaturase activities (Hastings et al, 2001). Initially, less attention was paid to fatty acid elongation, but recently genes specifically involved in the elongation of fatty acids in the HUFA biosynthetic pathway have been cloned and characterised from C. elegans (Beaudoin et al, 2000), the arachidonic acid-producing fungus Motierella alpina (Parker-Barnes et al, 2000), humans (Leonard et al, 2000b), rat (Inagaki et al, 2002) and zebrafish (Agaba et al, 2003).…”