Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species

Enhanced production of monounsaturated fatty acids (FA) derived from carbohydrate-enriched diets has been implicated in the development of obesity and insulin resistance. The FA elongases Elovl-5 and Elovl-6 are regulated by nutrient and hormone status, and have been shown using intact yeast and mammalian microsome fractions to be involved in the synthesis of monounsaturated FAs (MUFA). Herein, targeted knockdown and overexpression of Elovl-5 or Elovl-6 was used to determine their roles in de novo synthesis of specific MUFA species in mammalian cells. Treatment of rat insulinoma (INS)-1 cells with elevated glucose increased de novo FA synthesis and the ratio of MUFAs to saturated FAs. Elovl-5 knockdown decreased elongation of 16:1,n-7. Elovl-5 overexpression increased synthesis of 18:1,n-7; however, this increase was dependent on stearoyl-CoA desaturase–driven 16:1,n-7 availability. Knockdown of Elovl-6 decreased elongation of 16:0 and 16:1,n-7, resulting in accumulation of 16:1,n-7. Elovl-6 overexpression preferentially drove synthesis of 16:0 elongation products 18:0 and 18:1,n-9 but not 18:1,n-7. These findings demonstrate that coordinated induction of FA elongase and desaturase activity is required for balanced synthesis of specific n-7 versus n-9 MUFA species. Given the relative abundance of 16:0 to 16:1,n-7 and the specificity of Elovl-6 for 16:0, Elovl-6 is a major elongase for 18:1,n-9 production.     

Effect of dietary docosahexaenoic acid on biosynthesis of docosahexaenoic acid from alpha-linolenic acid in young rats

Docosahexaenoic acid (DHA), a crucial nervous system n-3 PUFA, may be obtained in the diet or synthesized in vivo from dietary alpha-linolenic acid (LNA). We addressed whether DHA synthesis is regulated by the availability of dietary DHA in artificially reared rat pups, during p8 to p28 development. Over 20 days, one group of rat pups was continuously fed deuterium-labeled LNA (d5-LNA) and no other n-3 PUFA (d5-LNA diet), and a second group of rat pups was fed a d5-LNA diet with unlabeled DHA (d5-LNA + DHA diet). The rat pups were then euthanized, and the total amount of deuterium-labeled docosahexaenoic acid (d5-DHA) (synthesized DHA) as well as other n-3 fatty acids present in various body tissues, was quantified. In the d5-LNA + DHA group, the presence of dietary DHA led to a marked decrease (3- to 5-fold) in the total amount of d5-DHA that accumulated in all tissues that we examined, except in adipose. Overall, DHA accretion from d5-DHA was generally diminished by availability of dietary preformed DHA, inasmuch as this was found to be the predominant source of tissue DHA. When preformed DHA was unavailable, d5-DHA and unlabeled DHA were preferentially accreted in some tissues along with a net loss of unlabeled DHA from other organs.     

Substrate preference in phosphatidylserine biosynthesis for docosahexaenoic acid containing species

Neuronal membranes contain high levels of phosphatidylserine (PS) and docosahexaenoic acid (22:6n-3, DHA). In this study, substrate preference in PS synthesis was determined to gain insight on the biochemical basis for concentrating PS in neuronal membranes where 22:6n-3 is highly enriched. We first established an in vitro assay method using unilamellar vesicles (LUV) of deuterium-labeled substrates and reversed-phase HPLC/electrospray ionization (ESI) mass spectrometry. The PS production by the incubation of deuterium-labeled substrate and microsomal fractions was monitored. We found that tissue-specific substrate preference exists in PS synthesis. Microsomes from the cerebral cortex synthesized PS from 18:0,22:6-PC most favorably among the PC substrates tested, followed by 18:0,22:5-PC, resulting in the PC substrate preference in the order of 18:0,22:6 > 18:0,22:5 > 18:0,20:4 = 18:0,18:1. Liver microsomes also preferred 18:0,22:6-PC as the substrate in PS synthesis but did not use 18:0,22:5-PC favorably. The 18:0,22:5-PC species was converted to PS at the similar extent as 18:0,20:4- or 18:0,18:1-PC species in the liver. Both brain and liver microsomes showed a preference for 18:0 over 16:0 as the sn-1 fatty acid. From these data it was deduced that preferential conversion of 18:0,22:6-PC to the corresponding PS species is at least partly responsible for concentrating PS in neuronal tissues where 22:6n-3 is particularly abundant. The distinctive preference for 18:0,22:5-PS observed with brain microsomes may help to maintain PS at a high level in the brain when 22:6n-3 is replaced by 22:5n-3 as in the case of n-3 fatty acid deficiency.     

Fatty acid elongases in mammals: their regulation and roles in metabolism

A significant amount of the fatty acids synthesized by the cytosolic enzyme complex fatty acid synthase (FAS) or taken up by the diet are further elongated into very long chain fatty acids (VLCFA) in a four-step reaction cycle by membrane-bound enzymes predominantly located in the endoplasmic reticulum. Members of the Elovl (elongation-of-very-long-chain-fatty acids) gene family encode for enzymes (elongases), which are believed to perform the first, regulatory, step (condensation) in the elongation cycle in mammals. The family of enzymes consists of at least six members in mouse and human, believed to carry out substrate-specific elongation with fatty acids of different lengths and degrees of unsaturation. The ability to synthesize VLCFA is a ubiquitous system found in different organs and cell types. However, VLCFAs seldom occur unesterified. Instead, they are joined either by an ester or amide linkage to a broad variety of different lipid species. VLCFA are most commonly found as building blocks in sphingolipids, although they are also important constituents of glycerophospholipids, triacylglycerols, sterol- and wax-esters. To generalize, the fatty acid elongases can be divided into two major groups: (a) enzymes which are suggested to be involved in the elongation of saturated and monounsaturated VLCFA (ELOVL1, 3 and 6) and (b) enzymes which are elongases of polyunsaturated fatty acids (PUFA) (ELOVL2, 4 and 5). All the elongases exhibit specific spatial and temporal expression. In this review, we will present and discuss the regulation of the mammalian fatty acid elongases and their potential role in lipid metabolism. We will consider both the biochemical functions of the proteins, as well as their role in a more physiological context.     

Evidence for the regulation of fatty acid utilization in human sperm by docosahexaenoic acid

The effects of stearic (18:0), linolenic (18:3), and docosahexaenoic (22:6) acids on palmitoyl coenzyme A (CoA) formation by a long-chain fatty acid:CoASH ligase (adenosine monophosphate) (E. C. 6.2.1.3-enriched fraction from human spermatozoa were studied. Both 18:0 and 18:3 were competitive inhibitors for palmitic (16:0) acid activation with Kis of 17.7 and 5.7 microM, respectively. In contrast, 22:6 was a noncompetitive inhibitor demonstrating a Ki of 9.5 microM. These data coupled with previous studies support the conclusion that 16:0, 18:0, and 18:3 and other saturated and unsaturated fatty acids are activated by the same ligase enzyme in sperm. Although the kinetics and interactions of 22:6 are unique compared to the other fatty acids found in sperm phospholipids, we cannot discern from our data if it is activated by a separate enzyme. We propose that 22:6, or a metabolite of 22:6, may regulate free fatty acid utilization in human sperm and that this hypothesis may provide an enzymatic explanation for the changes observed in phospholipid-bound fatty acids during the epididymal maturation of sperm.     

Cloning and functional characterisation of polyunsaturated fatty acid elongases of marine and freshwater teleost fish

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids are key to the biosynthesis of the highly unsaturated fatty acids, arachidonic, eicosapentaenoic and docosahexaenoic acids from linoleic and alpha-linolenic acids. A Mortierella alpina cDNA polyunsaturated fatty acid elongase sequence identified mammalian, amphibian, zebrafish and insect expressed sequence tags (ESTs) in GenBank. Consensus primers were designed in conserved motifs and used to isolate full length cDNA from livers of several fish species by Rapid Amplification of cDNA Ends (RACE). The amplified cDNAs encoded putative open reading frames (ORFs) of 288-294 amino acids that were highly conserved among the fish species. Heterologous expression in yeast, Saccharomyces cerevisiae, demonstrated that all of the ORFs encoded elongases with the ability to lengthen polyunsaturated fatty acid substrates with chain lengths from C18 to C22 and also monounsaturated fatty acids, but not saturated fatty acids. There were differences in the functional competence of the elongases from different fish species. Most of the fish elongases showed a pattern of activity towards different fatty acid substrates in the rank order C18>C20>C22, although the tilapia and turbot elongases had similar activity towards 18:4n-3 and 20:5n-3. The fish elongases generally showed greater activity or similar activities with n-3 than with n-6 homologues, with the exception of the cod enzyme which was more active towards n-6 fatty acids.     

The repertoire of desaturases and elongases reveals fatty acid variations in 56 eukaryotic genomes

The repertoire of biosynthetic enzymes found in an organism is an important clue for elucidating the chemical structural variations of various compounds. In the case of fatty acids, it is essential to examine key enzymes that are desaturases and elongases, whose combination determine the range of fatty acid structures. We systematically investigated 56 eukaryotic genomes to obtain 275 desaturase and 265 elongase homologs. Phylogenetic and motif analysis indicated that the desaturases consisted of four functionally distinct subfamilies and the elongases consisted of two subfamilies. From the combination of the subfamilies, we then predicted the ability to synthesize six types of fatty acids. Consequently, we found that the ranges of synthesizable fatty acids were often different even between closely related organisms. The reason is that, as well as diverging into subfamilies, the enzymes have functionally diverged within the individual subfamilies. Finally, we discuss how the adaptation to individual environments and the ability to synthesize specific metabolites provides some explanation for the diversity of enzyme functions. This study provides an example of a potent strategy to bridge the gap from genomic knowledge to chemical knowledge.     

Purification of the multienzyme complex for fatty acid oxidation from Pseudomonas fragi and reconstitution of the fatty acid oxidation system

The multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source. This complex exhibited enoyl-CoA hydratase [EC 4.2.1.17], 3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], 3-oxoacyl-CoA thiolase [EC 2.3.1.16], cis-3,trans-2-enoyl-CoA isomerase [EC 5.3.3.3], and 3-hydroxyacyl-CoA epimerase [EC 5.1.2.3] activities. The molecular weight of the native complex was estimated to be 240,000. Two types of subunits, with molecular weights of 73,000 and 42,000, were identified. The complex was composed of two copies each of the 73,000- and 42,000-Da subunits. The beta-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase. This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms. However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl-CoA was produced from 1 mol of linoleic or alpha-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of gamma-linolenic acid. These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative. When the epimerase was by-passed by the addition of 2,4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.     

Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization

The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LC-PUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ⁵- and Δ⁶-elongases of Marchantia polymorpha as well as the Δ⁶-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ⁹-, Δ⁶-C₁₈, and Δ⁵-C₂₀ LC-PUFAs, whereas only Δ⁹- and Δ⁶-C₁₈ LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C₂₀ fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C₂₀ substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFA-generating ELO enzymes.     

Cloning and sequencing of clustered genes involved in fatty acid biosynthesis from the docosahexaenoic acid-producing bacterium, Vibrio marinus strain MP-1

A cluster of genes involved in fatty acid biosynthesis (fab) was isolated from docosahexaenoic acid (DHA)-producing Vibrio marinus strain MP-1. This fab gene cluster included five genes highly homologous to the Escherichia coli counterparts, and their order in the cluster was the same with that of the E. coli fab gene cluster except that the latter included the additional fabH gene. These fab genes should be involved in early steps of DHA biosynthesis in V. marinus strain MP-1.     

Arachidonic acid induced coronary reactions and their inhibition by docosahexaenoic acid

The objective of the present study was to further investigate the influence exerted by docosahexaenoic acid (DHA) on the coronary reactions induced in isolated perfused hearts of rats and guinea pigs by bolus doses of arachidonic acid (AA). As in previous studies, we found that AA produced a coronary constriction followed by a longer lasting dilatation. The present data demonstrate that a 5-min infusion of DHA at 0.17-0.68 microM caused a concentration-dependent inhibition of the AA-induced constriction. The vasodilatation determined by AA was also depressed, but only after about 30 min of a sustained DHA infusion. The precursor of AA, linoleic acid (LA), was also infused for about 30 min, and like DHA it inhibited the coronary reactions induced by AA. LA is not converted into AA by the isolated heart, but like DHA, was probably incorporated into the cells of the coronary vascular compartment. It is known that LA, administered "in vivo" to mammals, is converted into AA and increases the production of eicosanoids, whereas DHA does not follow this metabolic pathway. The incorporation of these essential polyunsaturated fatty acids by the isolated perfused heart would inhibit the cyclooxygenase in the coronary vessel walls, interfering with the generation of vasomotor metabolites from AA. We postulate that the systemic administration of DHA, by inhibiting the synthesis of a constrictor metabolite, could be beneficial in reducing the damage due to microvascular constriction in myocardial ischaemia.     

Novel seed lipid phenotypes in combinations of mutants altered in fatty acid biosynthesis inArabidopsis

Summary The seed fatty acid (FA) composition of various single mutant combinations ofArabidopsis thaliana that affect FA biosynthesis has been examined. Double mutant combinations offae, a mutation affecting CIS elongation, and a series of four other FA biosynthetic mutants were synthesized. The four other single mutants were:fad2 andfad3, which are deficient in 181 and 182 desaturation, respectively;fab1, which is elevated in 160 and decreased in 181; andfab2, which is elevated in 180 and decreased in 181. The superimposition of two blocks in the FA biosynthetic pathway leads to dramatic changes in the FA content of the double mutants. The tenArabidopsis stocks analyzed to date (wild-type, five single mutants, and four double mutants) make seed oils with a wide range of FA compositions, and illustrate the diversity of oils it is possible to obtain from a single plant species.     

Antibacterial targets in fatty acid biosynthesis

The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for the development of new antibacterial agents. The extended use of the antituberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for antibacterial development. Differences in subcellular organization of the bacterial and eukaryotic multienzyme fatty acid synthase systems offer the prospect of inhibitors with host versus target specificity. Platensimycin, platencin, and phomallenic acids, newly discovered natural product inhibitors of the condensation steps in fatty acid biosynthesis, represent new classes of compounds with antibiotic potential. An almost complete catalog of crystal structures for the enzymes of the type II fatty acid biosynthesis pathway can now be exploited in the rational design of new inhibitors, as well as the recently published crystal structures of type I FAS complexes.     

Acyl carriers used as substrates by the desaturases and elongases involved in very long-chain polyunsaturated fatty acids biosynthesis reconstituted in yeast

The health benefits attributed to very long-chain polyunsaturated fatty acids and the long term goal to produce them in transgenic oilseed crops have led to the cloning of all the genes coding for the desaturases and elongases involved in their biosynthesis. The encoded activities have been confirmed in vivo by heterologous expression, but very little is known about the actual acyl substrates involved in these pathways. Using a Delta 6-elongase and front-end desaturases from different organisms, we have reconstituted in Saccharomyces cerevisiae the biosynthesis of arachidonic acid from exogenously supplied linoleic acid in order to identify these acyl carriers. Acyl-CoA measurements strongly suggest that the elongation step involved in polyunsaturated fatty acids biosynthesis is taking place within the acyl-CoA pool. In contrast, detailed analyses of lipids revealed that the two desaturation steps (Delta 5 and Delta 6) occur predominantly at the sn-2 position of phosphatidylcholine when using Delta 5- and Delta 6-desaturases from lower plants, fungi, worms, and algae. The specificity of these Delta 6-desaturases for the fatty acid acylated at this particular position as well as a limiting re-equilibration with the acyl-CoA pool result in the accumulation of gamma-linolenic acid at the sn-2 position of phosphatidylcholine and prevent efficient arachidonic acid biosynthesis in yeast. We confirm by using a similar experimental approach that, in contrast, the human Delta 6-desaturase uses linoleoyl-CoA as substrate, which results in high efficiency of the subsequent elongation step. In addition, we report that Delta 12-desaturases have no specificity toward the lipid polar headgroup or the sn-position.     

Identification of the peroxisomal beta-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid.

DHA (C22:6n-3) is an important PUFA implicated in a number of (patho)physiological processes. For a long time, the exact mechanism of DHA formation has remained unclear, but now it is known that it involves the production of tetracosahexaenoic acid (C24:6n-3) from dietary linolenic acid (C18:3n-3) via a series of elongation and desaturation reactions, followed by beta-oxidation of C24:6n-3 to C22:6n-3. Although DHA is deficient in patients lacking peroxisomes, the intracellular site of retroconversion of C24:6n-3 has remained controversial. By making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this article that peroxisomes, and not mitochondria, are involved in DHA formation by catalyzing the beta-oxidation of C24:6n-3 to C22:6n-3. Additional studies of fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, and of fibroblasts from l-bifunctional protein and sterol carrier protein X (SCPx) knockout mice, show that the main enzymes involved in beta-oxidation of C24:6n-3 to C22:6n-3 are SCOX, DBP, and both 3-ketoacyl-CoA thiolase and SCPx. These findings are of importance for the treatment of patients with a defect in peroxisomal beta-oxidation.