Biosynthesis of polyunsaturated fatty acids in lower eukaryotes (original) (raw)
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Reevaluation of the pathways for the biosynthesis of polyunsaturated fatty acids
Recent studies refute the commonly accepted, but untested, hypothesis that 7,10,13,16-22:4 and 7,10,13,16,19-22:5 are desaturated at position 4 by a microsomal acycl-CoA-independent desaturase. The synthesis of 4,7,10,13,16,19-22:6 occurs via the following reaction sequence: 4,7,10,13,16,19-22:6. The synthesis of 4,7,10,10,13,16-22:5 from 7,10,13,16-22:4 takes place via an analogous pathway. According to these pathways the 24-carbon acids that are made in the endoplasmic reticulum move to a site for partial beta-oxidation, 4,7,10,13,16-22:5 and 4,7,10,13,16,19-22:6, then move back to the endoplasmic reticulum where they are used as substrates for membrane lipid biosynthesis. The ability of fatty acid to serve as a substrate for continued peroxisomal beta-oxidation, versus its transfer out of peroxisomes for subsequent endoplasmic reticulum-associated esterification reactions, may be an important control for regulating membrane lipid fatty acid composition. Indeed, the revised pathways of polyunsaturated fatty acid biosynthesis imply that there is considerable intracellular movement endoplasmic reticulum. In addition, these revised pathways require that two 18-carbon and two 24-carbon acids are substrates for desaturation at position 6. Also, as linoleate and linolenate are metabolized, respectively, to 6,9,12,15,18-24:5 and 6,9,12,15,18,21-24:6, three n-6 acids and three n-3 acids are substrates for malonyl-CoA dependent chain elongation. It remains to be determined how many microsomal enzymes ancillary enzymes are expressed in tissues whose membrane lipids accumulate very long-chain polyunsaturated acids with up to 36 carbon atoms.
2000
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 g-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 dihomoGLA (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-...
Characterization of membrane-bound fatty acid desaturases
General physiology and biophysics, 2013
Membrane-bound desaturases play key role in metabolism of polyunsaturated fatty acids. Characterization of these enzymes and their genes is the first step in basic understanding of their proper functioning in living cells as well as in tailor-made preparation of highly-specific and highlyproductive strains of microorganisms interesting for applied biotechnology. It is also the crucial step in creation of transgenic agricultural crops with enhanced content of individual polyunsaturated fatty acids. Properties and applications of identified membrane-bound desaturases genes and enzymes are discussed in this review.
Structure and expression of fatty acid desaturases
Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1998
Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains. They are present in all groups of organisms, i.e., bacteria, fungi, plants and animals, and play a key role in the maintenance of the proper structure and functioning of biological membranes. The desaturases are characterized by the presence of three conserved histidine tracks which are presumed to compose the Fe-binding active centers of the enzymes. Recent findings on the structure and expression of different types of fatty acid desaturase in cyanobacteria, plants and animals are reviewed in this article. Roles of individual desaturases in temperature acclimation and principles of regulation of the desaturase genes are discussed. ß 1998 Elsevier Science B.V. All rights reserved.
Alternative route for the biosynthesis of polyunsaturated fatty acids in K562 cells
The Biochemical journal, 1993
K562 human leukaemia cells lack a significant delta 6-desaturase activity. However, they synthesize long-chain polyunsaturated fatty acids (PUFA) from linoleic (C18:2(9,12)) and linolenic (C18:3(9,12,15)) acids, by reactions involving a C2 chain elongation followed by a delta 5-desaturation step and, to some extent, a further elongation. The main products formed were separated by argentation t.l.c. and identified by g.l.c. as the uncommon fatty acids C20:3(5,11,14) and C20:4(5,11,14,17) respectively. These acids were also produced when cells were supplemented with C20:2(11,14) or C20:3(11,14,17) respectively. The presence of a delta 5-desaturase was further confirmed by using its corresponding normal substrates, C20:3(8,11,14) and C20:4(8,11,14,17), which led to C20:4(5,8,11,14) and C20:5(5,8,11,14,17) respectively. On the other hand, a high delta 9-desaturase activity, but no significant delta 4-desaturase activity, were detected in K562 cells. These results indicate the existence ...
Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis
The Journal of Lipid Research, 2004
In algae, the biosynthesis of docosahexaenoic acid (22:6 3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5 3; EPA) to 22:5 3, which is required as a substrate for the final ⌬ 4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana , from the fish Oncorhynchus mykiss , from the frog Xenopus laevis , and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens . The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana : one specific for the elongation of ( ⌬ 6-)C18-PUFAs and one specific for ( ⌬ 5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss , X. laevis , and C. intestinalis accepted both C18-and C20-PUFAs. By coexpression of the ⌬ 6-and ⌬ 5-elongases from T. pseudonana and O. tauri , respectively, with the ⌬ 5-and ⌬ 4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops. -Meyer, A.Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis.
The elongases of fatty acids (ELO) are essential for long chain polyunsaturated fatty acid (LC-PUFA) biosynthesis, and their activities depend on the substrates. The full length cDNA of Crassostrea angulata ELO (CaELO) was cloned by RACE PCR and its function was confirmed. The CaELO encodes a polypeptide of 309 amino acid residues, which containes a histidine box HXXHH motif conserved in all elongases and shares high similarity to the elongases of Chlamys nobilis and Octopus vulgaris. Phylogenetic analysis showed that the putative elongase was placed in the same group with ELOVL2 and ELOVL5, which have been demonstrated to be critical enzymes participating in the biosynthesis of PUFAs in vertebrates. When expressed in Saccharomyces cerevisiae, CaELO was able to elongate n-3 and n-6 PUFA substrates with chain lengths of C18 and C20, indicating that the CaELO had similar substrate specificities to vertebrate ELOVL5. CaELO had lower activity to elongate monounsaturated fatty acids, but had not activity to saturated fatty acids. Interestingly, the conversion rate of PUFAs depended on the length of carbon chain, the number of double bond, and n-3 / n-6 series in the species.
Biochemistry, 2000
To characterize the fatty acid desaturase produced by the fat-1 gene from the nematode Caenorhabditis elegans, the functional expression of this enzyme was effected in the yeast Saccharomyces cereVisiae. The GC-MS analysis of desaturated products derived from various fatty acids, including deuterium-labeled thia fatty acids supplied to growing cultures of transformed yeast, has defined the substrate requirements, regiochemistry, and cryptoregiochemistry of the enzyme. The desaturase acts on substrates of 16-20 carbons with a preference for ω-6 fatty acids, and its regioselectivity was confirmed to be that of an ω-3 desaturase. (ω-x refers to a double bond or desaturation between carbons x and x+1, counting from the methyl end of a fatty acid.) The primary deuterium kinetic isotope effects (KIEs) at C-15 and C-16 of a C18 fatty acid analogue were measured via competitive incubation experiments: While k H /k D at the ω-3 position was shown to be large (7.8 ( 0.4), essentially no KIE at the ω-2 position was observed (k H /k D ) 0.99 ( 0.04). This result indicates that ω-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. The results are discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring differing regioselectivities.
Saccharomyces cerevisiae is a valuable host for the expression and characterization of eukaryotic enzymes involved in polyunsaturated fatty acid (PUFA) biosynthesis, such as elongases and desaturases. The yeast allows a correct subcellular localization of these proteins, provides electron donors required by desaturases and is unable to synthesize PUFA that could interfere in the enzymes characterization. Unfortunately, S. cerevisiae incorporates very long chain PUFAs inefficiently, which could interfere in the characterization of enzymes using these substrates. Acyl-CoA synthe-tases (ACS) are involved in fatty acids uptake, and catalyze the synthesis of the corresponding CoA thioesters. ACS provides the substrates for elongases, acyl-CoA desaturases and acyl transferases. Transferases are required to synthesize phospholipids which in turn, are substrates for acyl-lipid desaturases. Expression in yeast of Trypanosoma brucei ACS1 notably improves the uptake of a wide variety of PUFA. Co-expression of ACS1 with Elo5 elongase from Leishmania major or Des4 desaturase from T. brucei showes, respectively, 2-and 5.6-fold increases in the uptake of the PUFA substrates and 2.4-and 3.5-fold increases in substrate conversion. It also allows to produce significant amount of Des4 desaturase product for further analysis, whereas it is obtained in trace amounts when the enzyme is expressed alone. Practical applications: In this report, the use of yeast strains expressing ACS1 is proposed as a useful tool in the characterization of polyunsaturated fatty acids desaturases and elongases. Furthermore, this model could be used for the production of nutraceutical PUFA.