Identification of a long-chain polyunsaturated fatty acid acyl-coenzyme A synthetase from the diatom Thalassiosira pseudonana

The draft genome of the diatom Thalassiosira pseudonana was searched for DNA sequences showing homology with long-chain acyl-coenzyme A synthetases (LACSs), since the corresponding enzyme may play a key role in the accumulation of health-beneficial polyunsaturated fatty acids (PUFAs) in triacylglycerol. Among the candidate genes identified, an open reading frame named TplacsA was found to be full length and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpLACSA, exhibited typical features of acyl-coenzyme A (acyl-CoA) synthetases involved in the activation of long-chain fatty acids. Feeding experiments carried out in yeast (Saccharomyces cerevisiae) transformed with the algal gene showed that TpLACSA was able to activate a number of PUFAs, including eicosapentaenoic acid and docosahexaenoic acid (DHA). Determination of acyl-CoA synthetase activities by direct measurement of acyl-CoAs produced in the presence of different PUFA substrates showed that TpLACSA was most active toward DHA. Heterologous expression also revealed that TplacsA transformants were able to incorporate more DHA in triacylglycerols than the control yeast.     

A spectrophotometric method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase

A mixture of Ti(IV) and 4-(2-pyridylazo)resorcinol was found to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide. The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide added. The reagent was successfully applied to the assay of free fatty acid in serum through the combined use of acyl-CoA synthetase and acyl-CoA oxidase. The latter enzyme produces H₂O₂. As a result, hydrogen peroxide was produced through the enzymatic oxidation of free fatty acid. It was possible to determine free fatty acid in 50 μl of serum at concentrations ranging from 0.02 to 1.5 mm. The coefficient of variation was less than 3% at concentrations ranging from 0.1 to 1.5 mm. In the present method, there is the advantage of minimal influence from reducible substances as well as greater simplicity and accuracy.     

Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin

The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.     

Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography-electrospray ionization tandem mass spectrometry

Acyl-CoAs have important role in fat and glucose metabolism of the cells. In this study we have developed an on-line HPLC-ESI-MS/MS method for determination of long-chain acyl-CoA compounds in rat liver samples. Six long-chain acyl-CoAs (C16:0, C16:1, C18:0, C18:1, C20:0 and C20:4) were separated with a C4 reversed-phase column using triethylamine acetate and acetonitrile gradient. Negative electrospray ionization is very suitable for acyl-CoA compounds and excellent MS/MS spectra for long-chain acyl-CoAs can be obtained. MS/MS method with an ion trap mass spectrometer makes it possible to identify and quantitate individual acyl-CoAs simultaneously. The method proved to be sensitive enough for determination of all compounds of interest using 0.4-0.7 g of tissue and was validated in the range of 0.1-15.0 pmol/microl.     

Molecular characterization of an Arabidopsis acyl-coenzyme a synthetase localized on glyoxysomal membranes

In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the beta-oxidation pathway, fatty acids need to be activated to acyl-coenzyme As (CoAs) by the enzyme acyl-CoA synthetase (ACS; EC 6.2.1.3). Here, we report the characterization of an Arabidopsis cDNA clone encoding for a glyoxysomal acyl-CoA synthetase designated AtLACS6. The cDNA sequence is 2,106 bp long and it encodes a polypeptide of 701 amino acids with a calculated molecular mass of 76,617 D. Analysis of the amino-terminal sequence indicates that acyl-CoA synthetase is synthesized as a larger precursor containing a cleavable amino-terminal presequence so that the mature polypeptide size is 663 amino acids. The presequence shows high similarity to the typical PTS2 (peroxisomal targeting signal 2). The AtLACS6 also shows high amino acid identity to prokaryotic and eukaryotic fatty acyl-CoA synthetases. Immunocytochemical and cell fractionation analyses indicated that the AtLACS6 is localized on glyoxysomal membranes. AtLACS6 was overexpressed in insect cells and purified to near homogeneity. The purified enzyme is particularly active on long-chain fatty acids (C16:0). Results from immunoblot analysis revealed that the expression of both AtLACS6 and beta-oxidation enzymes coincide with fatty acid degradation. These data suggested that AtLACS6 might play a regulatory role both in fatty acid import into glyoxysomes by making a complex with other factors, e.g. PMP70, and in fatty acid beta-oxidation activating the fatty acids.     

Fatty acid export from the chloroplast. Molecular characterization of a major plastidial acyl-coenzyme A synthetase from Arabidopsis

Acyl-coenzyme A (CoA) synthetases (ACSs, EC 6.2.1.3) catalyze the formation of fatty acyl-CoAs from free fatty acid, ATP, and CoA. Essentially all de novo fatty acid synthesis occurs in the plastid. Fatty acids destined for membrane glycerolipid and triacylglycerol synthesis in the endoplasmic reticulum must be first activated to acyl-CoAs via an ACS. Within a family of nine ACS genes from Arabidopsis, we identified a chloroplast isoform, LACS9. LACS9 is highly expressed in developing seeds and young rosette leaves. Both in vitro chloroplast import assays and transient expression of a green fluorescent protein fusion indicated that the LACS9 protein is localized in the plastid envelope. A T-DNA knockout mutant (lacs9-1) was identified by reverse genetics and these mutant plants were indistinguishable from wild type in growth and appearance. Analysis of leaf lipids provided no evidence for compromised export of acyl groups from chloroplasts. However, direct assays demonstrated that lacs9-1 plants contained only 10% of the chloroplast long-chain ACS activity found for wild type. The residual long-chain ACS activity in mutant chloroplasts was comparable with calculated rates of fatty acid synthesis. Although another isozyme contributes to the activation of fatty acids during their export from the chloroplast, LACS9 is a major chloroplast ACS.     

Beta-oxidation of long-chain fatty acids by human fibroblasts: evidence for a novel long-chain acyl-coenzyme A dehydrogenase.

Fibroblasts from patients with long-chain acyl-CoA dehydrogenase deficiency were found to oxidize [1-14C]linoleate at an average rate of 60% of normal but [9,10(n)-3H]myristate at an average rate of only 37% of normal, a relationship reverse from that predicted by the chain-length specificities of the three known straight-chain mitochondrial acyl-CoA dehydrogenases. The residual long-chain beta-oxidative activity was found to be mitochondrial and associated with the accumulation of tetradecadienoate (C14:2w6) when the mutant fibroblasts were incubated with 100 mumol/L linoleate (C18:2w6) or eicosadienoate (C20:2w6). The results suggest the presence in human fibroblasts of a novel acyl-CoA dehydrogenase with activity toward 15 to 20 carbon-length fatty acids.     

Mutations in LACS2, a long-chain acyl-coenzyme A synthetase, enhance susceptibility to avirulent Pseudomonas syringae but confer resistance to Botrytis cinerea in Arabidopsis

We identified an Arabidopsis (Arabidopsis thaliana) mutant, sma4 (symptoms to multiple avr genotypes4), that displays severe disease symptoms when inoculated with avirulent strains of Pseudomonas syringae pv tomato, although bacterial growth is only moderately enhanced compared to wild-type plants. The sma4 mutant showed a normal susceptible phenotype to the biotrophic fungal pathogen Erysiphe cichoracearum. Significantly, the sma4 mutant was highly resistant to a necrotrophic fungal pathogen, Botrytis cinerea. Germination of B. cinerea spores on sma4 mutant leaves was inhibited, and penetration by those that did germinate was rare. The sma4 mutant also showed several pleiotropic phenotypes, including increased sensitivity to lower humidity and salt stress. Isolation of SMA4 by positional cloning revealed that it encodes LACS2, a member of the long-chain acyl-CoA synthetases. LACS2 has previously been shown to be involved in cutin biosynthesis. We therefore tested three additional cutin-defective mutants for resistance to B. cinerea: att1 (for aberrant induction of type three genes), bodyguard, and lacerata. All three displayed an enhanced resistance to B. cinerea. Our results indicate that plant cutin or cuticle structure may play a crucial role in tolerance to biotic and abiotic stress and in the pathogenesis of B. cinerea.     

Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism

Long-chain fatty acid amides are cell-signaling lipids identified in mammals and, recently, in invertebrates, as well. Many details regarding fatty acid amide metabolism remain unclear. Herein, we demonstrate that Drosophila melanogaster is an excellent model system for the study long-chain fatty acid amide metabolism as we have quantified the endogenous levels of N-acylglycines, N-acyldopamines, N-acylethanolamines, and primary fatty acid amides by LC/QTOF-MS. Growth of D. melanogaster on media supplemented with [1-¹³C]-palmitate lead to a family of ¹³C-palmitate-labeled fatty acid amides in the fly heads. The [1-¹³C]-palmitate feeding studies provide insight into the biosynthesis of the fatty acid amides.     

Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation

Post-germinative growth of oilseeds is dependent on the breakdown of the stored lipid reserves. Long-chain acyl-CoA synthetase activities (LACS) are critically involved in this process by activating the released free fatty acids and thus feeding the beta-oxidation cycle in glyoxysomes. Here we report on the identification of two LACS genes, AtLACS6 and AtLACS7 from Arabidopsis thaliana coding for peroxisomal LACS proteins. The subcellular localization was verified by co-expression studies of spectral variants of the green fluorescent protein (GFP). While AtLACS6 is targeted by a type 2 (PTS2) peroxisomal targeting sequence, for AtLACS7 a functional PTS1 as well as a PTS2 could be demonstrated. Possible explanations for this potentially redundant targeting information will be discussed. Expression studies of both genes revealed a strong induction 1 day after germination resembling the expression pattern of other genes involved in beta-oxidation. Analysis of the substrate specificities of the two LACS proteins demonstrated enzymatic activity for both enzymes with the whole spectrum of fatty acids found in stored lipid reserves. These results suggest that both LACS proteins might have overlapping functions and are able to initiate beta-oxidation in plant peroxisomes.     

Molecular characterization of a rabbit long-chain fatty acyl CoA synthetase that is highly expressed in the vascular endothelium

The formation of coenzyme A thioesters from long-chain fatty acids represents a metabolic branch point. We have isolated, cloned and sequenced a long-chain fatty acyl CoA synthetase (LCFACoAS) that is localized to the endothelium of rabbit heart and aorta. Immunofluoresence and in situ hybridization studies show intense staining of the intimal layer of the aorta and coronary vessels. The microvessels, including the capillaries, of the coronary circulation also show intense immunofluoresence. The enzyme shares only about 30% to 70% homology with the primary amino acid sequence of the other known LCFACoAS. There is a region of 44 amino acids at the carboxy terminus, which is unique to the vascular enzyme. This domain contains the most hydrophobic region of the molecule, indicating that it may function as a membrane anchoring site. These results suggest that this LCFACoAS represents a novel isoform, whose functional significance remains to be determined.     

Identity of long-chain acyl-coenzyme A synthetase of microsomes, mitochondria, and peroxisomes in rat liver

The identity of long-chain acyl-CoA synthetase in microsomes, mitochondria, and peroxisomes of rat liver was examined by using the antibody raised against a purified preparation of the microsomal enzyme. The enzyme activities of these three organelles and the purified microsomal enzyme were titrated by the antibody in a very similar fashion when the activity was measured in terms of palmitoyl-CoA synthetase activity. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitates and by Western blot analysis that the enzymes of all three organelles consisted of a polypeptide with the same molecular weight as that of the purified enzyme, and that the specific enzyme activity of the antigenic protein in all three subcellular compartments was nearly the same. The presence of other palmitoyl-CoA synthetase activity in these organelles could not be confirmed. Immunocytochemical study to locate the antigenic site with protein A-gold complex showed that the gold particles were closely associated with the membranes of these organelles. The cell-free translation product in a rabbit reticulocyte lysate protein-synthesizing system and the subunit of the mature enzyme labeled with [35S]methionine in the liver slices exhibited the same mobility as the subunit of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme in microsomes, mitochondria, and peroxisomes was labeled at nearly the same rate when the liver slices were incubated with [35S]methionine.     

Isolation and characterization of a long-chain acyl-coenzyme A synthetase encoding gene from the marine microalga Nannochloropsis oculata

Acyl-coenzyme A synthetases (ACSs) are associated with the anabolism and catabolism of fatty acids and play fundamental roles in various metabolic pathways. The cDNA of long-chain acyl-coenzyme A synthetase (LACS), one of the ACSs, was isolated from Nannochloropsis oculata and named as NOLACS. The predicted amino acid sequence was highly similar to LACSs of other species. NOLACS encodes a long-chain acyl-coenzyme A synthetase; it recovered the function of LACS in Saccharomyces cerevisiae YB525 (a LACS-deficient yeast strain). The substrate specificity of the enzyme was also assayed in yeast. It was found that NOLACS can activate saturated fatty acids (C12:0, C14:0, C16:0, and C18:0) and some unsaturated fatty acids (C18:2Δ^{9, 12} and C20:2Δ^{11, 14}) with a preference for long-chain fatty acids. Our findings will provide a deep understanding of CoA-dependent fatty acid activation and also make some contribution to understanding the metabolic pathways of lipids in Nannochloropsis. These findings will also facilitate studies on the regulation of gene expression and genetic modification of fatty acid synthesis and storage of N. oculata.     

Non-esterified long-chain fatty acid metabolism in fed sheep at rest and during exercise

The role of circulating, non-esterified, long-chain fatty acids (NEFA) as a source of energy for the whole animal and skeletal muscle was investigated in fed non-pregnant sheep at rest and during exercise. Infusion of tracer quantities of [1-14C]oleic or [1-14C]stearic acid was combined with the use of arteriovenous difference studies on fed sheep at rest or during a 2 h period of exercise on a belt treadmill moving at 4.5 km h-1. At rest all parameters of NEFA metabolism indicated a minimal role for oxidation. Thus the concentration in plasma (0.07 +/- 0.01 mmol l-1), entry rate (0.08 +/- 0.02 mmol h-1 kg-1 body wt), contribution to whole animal oxidation (1.2 +/- 0.3%) and utilization of NEFA by skeletal muscle (0.046 +/- 0.008 mmol h-1 kg-1 muscle) were all low. Exercise prompted a shift to lipolysis and accordingly the above parameters increased markedly some 13-24-fold. The circulating concentration of ketone bodies showed only a small increase during exercise and consequently the role of ketone bodies as an energy source during exercise was minimal. Glucose utilization by skeletal muscle was considerable in animals at rest and it represented the most significant potential fuel of skeletal muscle. Exercise resulted in a sustained increase of 3-4-fold in the utilization of glucose by skeletal muscle. Thus the traditional view that NEFA and not glucose is a predominant fuel of skeletal muscle of fed sheep should be appraised.