Domain swapping localizes the structural determinants of regioselectivity in membrane-bound fatty acid desaturases of Caenorhabditis elegans

Most fatty acid desaturases are members of a large superfamily of integral membrane, O(2)-dependent, iron-containing enzymes that catalyze a variety of oxidative modifications to lipids. Sharing a similar primary structure and membrane topology, these enzymes are broadly categorized according to their positional specificity or regioselectivity, which designates the preferred position for substrate modification. To investigate the structural basis of regioselectivity in membrane-bound desaturases, the Caenorhabditis elegans omega-3 (FAT-1) and "Delta12" (FAT-2) desaturases were used as a model system. With the use of unnatural substrates, the regioselectivity of C. elegans FAT-2 was clearly defined as nu+3, i.e. it "measures" three carbons from an existing double bond. The structural basis for nu+3 and omega-3 regioselectivities was examined through construction and expression of chimeric DNA sequences based on FAT-1 and FAT-2. Each sequence was divided into seven domains, and chimeras were constructed in which specific domains were replaced with sequence from the other desaturase. When tested by expression in yeast using exogenously supplied substrates, chimeric sequences were found in which domain swapping resulted in a change of regioselectivity from nu+3 to omega-3 and vice versa. In this way, the structural determinants of regioselectivity in FAT-1 and FAT-2 have been localized to two interdependent regions: a relatively hydrophobic region between the first two histidine boxes and the carboxyl-terminal region.     

Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase (FAT-1) expressed in Saccharomyces cerevisiae

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 omega-6 fatty acids, and its regioselectivity was confirmed to be that of an omega-3 desaturase. (omega-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 omega-3 position was shown to be large (7.8 +/- 0.4), essentially no KIE at the omega-2 position was observed (k(H)/k(D) = 0.99 +/- 0.04). This result indicates that omega-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.     

Monohydroxylated fatty acid substrate specificity of human leukocyte 5-lipoxygenase and omega-hydroxylase.

Various monohydroxylated fatty acids were synthesized from eicosapolyenoic acids, namely arachidonic (20:4 omega-6), timnodonic (20:5 omega-3), dihomogammalinolenic (20:3 omega-6) and mead (20:3 omega-9) acids. 12-Hydroxy derivatives, as well as 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), were produced with platelets as the enzyme source, and 15-hydroxy derivatives were produced by soya bean lipoxygenase treatment. Each monohydroxylated fatty acid was incubated with human leukocytes in the presence or absence of the calcium ionophore A23187, and dihydroxylated products were analysed by h.p.l.c. 12-Hydroxy derivatives of 20:4 omega-6, 20:5 omega-3 and 20:3 omega-9 were similarly oxygenated by both the 5-lipoxygenase and the omega-hydroxylase. As expected, the 12-hydroxy derivative of 20:3 omega-6 was not a substrate for 5-lipoxygenase, but surprisingly, omega-6 oxygenated products, like 15-OH-20:4 or HHT, were not converted by the enzyme, although being potential substrates because of the presence of two double bonds at C-5 and C-8. omega-6 oxygenated derivatives were also poorly converted by leukotriene B4 omega-hydroxylase, a cytochrome P-450-dependent enzyme. It is concluded that both leukocyte 5-lipoxygenase and omega-hydroxylase exhibit a substrate specificity towards monohydroxylated fatty acids with respect to their double bonds and/or the carbon position of the alcohol function.     

Caenorhabditis elegans Delta12-desaturase FAT-2 is a bifunctional desaturase able to desaturate a diverse range of fatty acid substrates at the Delta12 and Delta15 positions

Caenorhabditis elegans FAT-2 has been characterized as fatty acid Δ12-desaturase able to desaturate C16 and C18 fatty acids. However, in this report we show that when expressed in yeast cells this enzyme can also catalyze Δ15 desaturation. This results in the production of both linoleic acid (ω6 C18:2Δ9,12) and linolenic acid (ω3 C18:3Δ9,12,15) from oleic acid (C18:1Δ9) substrate, and hexadecadienoic acid (ω4 C16:2Δ9,12) and hexadecatrienoic acid (ω1 C16:3Δ9,12,15) from palmitoleic acid (C16:1Δ9) substrate. In addition, this enzyme can also produce C14:2Δ9,12, C15:2Δ9,12, C17:2Δ9,12, and C18:4Δ6,9,12,15 when C14:1Δ9, C15:1Δ9, C17:1Δ9, and C18:3Δ6,9,12 substrates are available in yeast cells. Mass spectrometry analysis of 2,4-dimethyloxazoline modification of fatty acid methyl esters confirms the positions of all newly formed double bonds. These results indicate that when expressed in yeast the C. elegans Δ12-desaturase CeFAT-2 shows a characteristic of a bifunctional Δ12/Δ15-desaturase and has a great deal of elasticity with respect to fatty acid chain length in being able to accept fatty acids ranging from C14 to C18. Interestingly, despite possessing a bifunctional Δ12/Δ15 desaturation activity, phylogenetic analysis suggests that C. elegans Δ12-desaturase CeFAT-2 might have arisen independently from other reported dual Δ12/Δ15-desaturases from fungi and protozoa.     

Endoplasmic oleoyl-PC desaturase references the second double bond

The regiospecificity for the gene product of fad2,(1) the microsomal oleoyl-PC desaturase from higher plants, differs from some previous suggestions. Rather than only referencing the carboxyl group (a Delta(12) desaturase) or the methyl terminus (an omega-6 desaturase), this desaturase locates the second double bond in its substrates by first referencing the existing double bond. This specificity was demonstrated for the oleoyl-PC desaturase cDNA from the developing seeds of peanut (Arachis hypogaea L) expressed in yeast (Saccharomyces cerevisae). The expressed enzyme was capable of desaturating monounsaturated fatty acyl groups in membrane lipids. Endogenous palmitoleate was desaturated to cis, cis 9,12 hexadecadienoate (9(Z)12(Z)C16:2), endogenous oleate to linoleate (9(Z)12(Z) octadecadienoate), and cis 10-nonadecenoate (provided as a supplement in the growth medium) to 10(Z)13(Z)C19:2. The rule, Delta(x+3) where x=9 is the double bond location in the substrate, best describes the consistent placement of the second double bond in the above monounsaturated substrates for the oleoyl-PC desaturase of higher plants.     

Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the omega-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast. The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16-20 carbons with a preference for omega-6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of omega-3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C(18) fatty acid analogue were measured via competitive incubation experiments. Whereas k(H)/k(D) at the omega-3 position was shown to be large, essentially no kinetic isotope effect at the omega-2 position was observed for the plant or the nematode enzymes. These results indicate that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.     

Delta-8 desaturation activity varies among fatty acyl desaturases of teleost fish: high activity in delta-6 desaturases of marine species

The benefits of dietary fish and fish oil are derived from n-3 long-chain polyunsaturated fatty acids (LC-PUFA) that have beneficial effects in a range of human diseases and pathologies such as cardiovascular and other inflammatory disorders, neural development and neurological pathologies. The precursor of n-3 LC-PUFA, 18:3n-3 does not have the same beneficial effects prompting interest in the pathways of endogenous synthesis of LC-PUFA in vertebrates. The LC-PUFA biosynthesis pathway classically involves Δ6 and Δ5 fatty acyl desaturases (Fad), but it was recently shown that Δ6 Fad in mammals also displayed Δ8 activity demonstrating a possible alternative "Δ8-pathway" for the synthesis of LC-PUFA. Our primary hypothesis was that Δ8 desaturase activity would be a common feature of vertebrate Δ6 Fads, and so the aim of the present study was to determine the ability of teleostei Fads for Δ8 desaturation activity. To this end, cDNAs for Fads from a range of freshwater, diadromous and marine teleost fish species were assayed for Δ8 activity in the heterologous yeast expression system. In summary, the present study has demonstrated that Δ8 desaturation activity was also a characteristic of fish orthologs, although the activity varied notably between freshwater/diadromous and marine fish species, with the latter possessing Fads2-like proteins with Δ8 activity far higher than mammalian FADS2. The data showed that, generally, the fish Fad are technically υ-3 desaturases, with new double bonds introduced 3C beyond a pre-existing double bond. However, the ability of zebrafish and rabbitfish Fads, previously characterised as Δ6/Δ5 bifunctional desaturases, to introduce non-methylene interrupted double bonds in 20:3n-3 and 20:2n-6 suggested that a novel combination of regioselectivity modes operates within these enzymes.     

The use of deoxyfluoro-d-galactopyranoses in a study of yeast galactokinase specificity

1. 2-Deoxy-2-fluoro-d-galactose, 3-deoxy-3-fluoro-d-galactose, 4-deoxy-4-fluoro-d-galactose, 6-deoxy-6-fluoro-d-galactose and 2-deoxy-d-lyxo-hexose are substrates for yeast galactokinase. 2. The variation in K(m) values for the d-hexose derivatives was not associated with a variation in the value of K(m) for MgATP(2-) indicating that the binding of MgATP(2-) is not modified by the binding of the sugar substrate. 3. Donated H bonds from OH-3, OH-4 and OH-6 and an accepted H bond to OH-2 of the d-hexose are important for the binding of the sugar substrate to galactokinase. 4. Yeast galactokinase exhibits similar kinetics to the galactokinase from Escherichia coli and operates by a similar random sequential mechanism. 5. 4-Deoxy-4-fluoro-d-glucose was neither a substrate for nor an inhibitor of yeast galactokinase.     

Reductive biotransformations of organic compounds by cells or enzymes of yeast.

Saccharomyces cerevisiae catalyses the asymmetric reductive biotransformation of a variety of compounds containing a carbonyl group or carbon-carbon double bond. Oxidoreductases participating in these reactions which have commercial potential in biotransformation processes are likely to have relatively broad substrate specificity. Important carbonyl reductases falling into this category include YADH- and yeast NADP-dependent beta-ketoester reductases. The enoyl reductase component of the FAS complex may have a role in asymmetric yeast reduction of carbon-carbon double bonds of unnatural substrates. Other nicotinamide-requiring oxidoreductases of yeast are also surveyed to rationalize observed biotransformations of whole yeast cells in terms of specific enzymes. Genetic and protein engineering may enable enzymes to be tailored to accept new substrates. A greater understanding of the enzymes and reactions involved will facilitate further optimization and exploitation of these catalytic systems in industrial processes.     

Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: formation of a novel metabolite by a female-specific P-450

Previous studies demonstrated that liver microsomes from untreated rats catalyze the omega, omega-1, and omega-2 hydroxylation of prostaglandins [K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489]. The current study examined the regioselectivity of hydroxylation of PGE1 and PGE2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH-P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE1, RLM 5 catalyzed solely the omega-1 hydroxylation and 5a catalyzed primarily the omega-1 and little omega and omega-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE1 and PGE2 at the omega-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is omega-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE1 hydroxylation demonstrated that the affinity of f4 for PGE1 is approximately 100-fold higher than that of RLM 5; the Km values for f4, monitoring 19- and 17-hydroxylation of PGE1, were about 10 microM. Surprisingly, cytochrome b5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE2 by RLM 5 was at the omega, omega-1, and omega-2 sites, demonstrating a lesser regioselectivity than with PGE1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b5 requirement.     

A palmitoyl-CoA-specific delta9 fatty acid desaturase from Caenorhabditis elegans

Biosynthesis of polyunsaturated fatty acids in C. elegans is initiated by the introduction of a double bond at the delta9 position of a saturated fatty acid. We identified three C. elegans fatty acid desaturase genes related to the yeast delta9 desaturase OLE1 and the rat stearoyl-CoA desaturase SCD1. Heterologous expression of all three genes rescues the fatty acid auxotrophy of the yeast delta9 desaturase mutant ole1. Examination of the fatty acid composition of the transgenic yeast reveals striking differences in the substrate specificities of these desaturases. Two desaturases, FAT-6 and FAT-7, readily desaturate stearic acid (18:0) and show less activity on palmitic acid (16:0). In contrast, the other desaturase, FAT-5, readily desaturates palmitic acid (16:0), but shows nearly undetectable activity on the common delta9 substrate stearic acid. This is the first report of a palmitoyl-CoA-specific membrane fatty acid desaturase.     

Substrate specificity of tyrosyl-DNA phosphodiesterase I (Tdp1)

Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes 3'-phosphotyrosyl bonds to generate 3'-phosphate DNA and tyrosine in vitro. Tdp1 is involved in the repair of DNA lesions created by topoisomerase I, although the in vivo substrate is not known. Here we study the kinetic and binding properties of human Tdp1 (hTdp1) to identify appropriate 3'-phosphotyrosyl DNA substrates. Genetic studies argue that Tdp1 is involved in double and single strand break repair pathways; however, x-ray crystal structures suggest that Tdp1 can only bind single strand DNA. Separate kinetic and binding experiments show that hTdp1 has a preference for single-stranded and blunt-ended duplex substrates over nicked and tailed duplex substrate conformations. Based on these results, we present a new model to explain Tdp1/DNA binding properties. These results suggest that Tdp1 only acts upon double strand breaks in vivo, and the roles of Tdp1 in yeast and mammalian cells are discussed.     

Formation of trans-4-hydroxy-2-nonenal- and other enal-derived cyclic DNA adducts from omega-3 and omega-6 polyunsaturated fatty acids and their roles in DNA repair and human p53 gene mutation

The cyclic 1,N(2)-propanodeoxyguanosine adducts, derived from alpha,beta-unsaturated aldehydes or enals, including acrolein (Acr), crotonaldehyde (Cro), and trans-4-hydroxy-2-nonenal (HNE), have been detected as endogenous DNA lesions in rodent and human tissues. Collective evidence has indicated that the oxidative metabolism of polyunsaturated fatty acids (PUFAs) is an important pathway for endogenous formation of these adducts. In a recent study, we examined the specific role of different types of fatty acids, omega-3 and omega-6 PUFAs, in the formation of cyclic adducts of Acr, Cro, and HNE. Our studies showed that the incubation of deoxyguanosine 5'-monophosphate with omega-3 or omega-6 fatty acids under oxidative conditions in the presence of ferrous sulfate yielded different amounts of Acr, Cro, and HNE adducts, depending on the types of fatty acids. We observed that Acr- and Cro-dG adducts are primarily formed from omega-3, and the adducts derived from longer chain enals, such as HNE, were detected exclusively from omega-6 fatty acids. Acr adducts are also formed from omega-6 fatty acids, but to a lesser extent; the yields of Acr adducts are proportional to the number of double bonds present in the PUFAs. Two previously unknown cyclic adducts, one from pentenal and the other from heptenal, were detected as products from omega-3 and omega-6 fatty acids, respectively. Because omega-6 PUFAs are known to be involved in the promotion of tumorigenesis, we investigated the role of HNE adducts in p53 gene mutation by mapping the HNE binding to the human p53 gene with UvrABC nuclease and determined the formation of HNE-dG adducts in the gene. The results showed that HNE-dG adducts are preferentially formed in a sequence-specific manner at the third base of codon 249 in the p53 gene, a mutational hotspot in human cancers. The DNA repair study using plasmid DNA containing HNE-dG adducts as a substrate in HeLa cell extracts showed that HNE adducts are readily repaired, and that nucleotide excision repair appears to be a major pathway involved. Together, results of these studies provide a better understanding of the involvement of different PUFAs in DNA damage and their possible roles in tumorigenesis.     

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Candida albicans contains 10 putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6x(His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, omega-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (omega-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d(3)-dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the omega- to the (omega-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The regioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYP4A family of enzymes, does not involve covalent binding of the heme to the protein.     

Activity of human Delta5 and Delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.

Yeast co-expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n-3 and n-6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Delta5 desaturase is active on 20:3n-6 and 20:4n-3. Endogenous Delta6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n-6 or 20:5n-3 which, through two sequential elongations, produced 24:4n-6 and 24:5n-3, respectively. Overall, our data suggest that a single human Delta6 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.     

Incorporation of 14C-labelled polyunsaturated fatty acids by juvenile turbot, Scophthalmus maximus (L.) in vivo

The ability of juvenile turbot, Scophthalmus maximus (L.), to elongate and desaturate various polyunsaturated fatty acids (PUFA) was examined in relation to their lipid composition. Triacylglycerols were the most abundant lipid class present in the fish and phosphatidylcholine was the predominant phospholipid. In all lipid classes examined the levels of (n-3) PUFA exceeded that of (n-6) PUFA. 18C PUFA were minor components in comparison with 20:5(n-3) and 22:6(n-3). 20:4(n-6) was present in highest concentration in phosphatidylinositol in which it accounted for 16.9% of the fatty acids. When the fish were injected with either ¹⁴C-labelled 18:2(n-6), 18:3(n-3), 20:4(n-6), 20:5(n-3) or 22:6(n-3) the highest percentage recovery of radioactivity (69%) in body lipid was observed with 22:6(n-3). With all labelled substrates free fatty acids contained only a small proportion of the total recovered radioactivity whereas triacylglycerols were highly labelled. Phosphatidylcholine/sphingomyelin was the most highly labelled polar lipid fraction. With ¹⁴C-20:4(n-6) as injected substrate, 23.2% of the radioactivity recovered in total lipid was present in phosphatidylinositol in comparison with less than 6% with the other substrates. Only small proportions of radioactivity from ¹⁴C-18:2(n-6) and ¹⁴C-18:3(n-3) were recovered in the 20 and 22C fatty acids of triacylglycerols and total polar lipid. With ¹⁴C-20:5(n-3) as substrate, 27 and 33% of the total radioactivity recovered in the fatty acids of triacylglycerols and polar lipids respectively was present in 22C fatty acids. The corresponding values for ^l4C-20:4(n-6) as substrate were 19 and 18%. The results confirm the limited capacity of turbot to convert 18C PUFA to longer chain PUFA but demonstrate their ability to synthesize 22C PUFA from 20C PUFA. They also suggest a small but specific requirement for 20:4(n-6).     

Natural competence in strains of Actinobacillus pleuropneumoniae

The fatty acid (FA) profiles of myxobacteria contain FA species with double bonds at the Δ⁵ and Δ¹¹ positions, the latter being rather unusual among bacteria. Despite this knowledge, the mechanism for introduction of these double bonds has never been described before in myxobacteria. Searches for candidate genes in the genome of the model organism Myxococcus xanthus revealed 16 genes, which have been annotated as FA desaturases. However, due to redundant substrate specificity, functional analyses of these enzymes by construction of inactivation mutants did not lead to the identification of their function or substrate specificity. Therefore, we elucidated the regioselectivity of the desaturation reactions by heterologous expression of eight desaturases from M. xanthus in Pseudomonas putida and thus could prove five of them to be indeed active as desaturases, with three (MXAN_1742, MXAN_3495 and MXAN_5461) and two (MXAN_0317 and MXAN_6306) acting as Δ⁵ and Δ¹¹ desaturases, respectively. This is the first report about the heterologous expression and regioselectivity of FA desaturases in myxobacteria.     

Enzymatic cleavage of the epsilon-peptide bond in alpha- and epsilon-substituted glycyl- and phenylalanyl-lysine peptides

Lysine peptides, X-Lys-OH (Formula: see text) were synthesized, following classic or non-classic routes. Some bacterial and mammalian enzymes, endo- and exo-peptide hydrolases of the enzyme nomenclature type EC 3.4., were tested for their ability to split the epsilon-peptide bond in the above substrates. Kinetic constants (Km,kcat) were evaluated with leucine aminopeptidase from hog kidney and eye lens with aminopeptidase I from yeast. Aminopeptidase M (hog pancreas) and hog intestinal aminopeptidase were additionally examined for their Ki values with the above substrates in comparison to the classic protease substrate leucine p-nitroanilide. Especially the intestinal mucosa hydrolases are shown to be efficient in cleaving epsilon-peptide bonds.     

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C_18/20 polyunsaturated fatty acid (PUFA) substrates, but not C₂₂ PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.