Analysis of the condensation step in elongation of very-long-chain saturated and tetraenoic fatty acyl-CoAs in swine cerebral microsomes

The condensation products in the elongation of exogenous arachidoyl-CoA (20:0-CoA) and endogenous fatty acids in adult swine cerebral microsomes were isolated and purified by using HPLC and a radioanalyzer. A saponification product of the condensation reaction of 20:0-CoA with malonyl-CoA was identified by gas chromatography-mass spectrometry as 2-heneicosanone (21:0-2-one). The endogenous substrates (16:0-CoA and 20:4-CoA) were likewise identified as 2-heptadecanone (17:0-2-one) and 2-heneicosatetraenone (21:4-2-one). Quantitative analysis of condensation activity was performed using electron-impact mass fragmentography. A characteristic fragment ion (m/z 59) of 21:0-2-one was used to estimate the condensation activity for 20:0-CoA, and fragment ions at m/z 58 and 80 were monitored for the endogenous substrates (16:0-CoA and 20:4-CoA, respectively). The molecular ion for each product was detected using chemical ionization. A comparative study of the condensation of 20:0-CoA and endogenous substrates was carried out for microsomes obtained from white matter, gray matter, and isolated neuronal cells; the activity for 20:0-CoA was significantly lower in gray matter and neuronal cells than in white matter, whereas the activity for endogenous substrates was almost the same for microsomes obtained from gray and white matter. This result suggests that the condensation enzyme for 20:0-CoA may be different from that for endogenous 16:0-CoA or 20:4-CoA in swine cerebral microsomes.     

Inhibitory effect of very-long-chain monounsaturated fatty-acyl-CoAs on the elongation of long-chain fatty acid in swine cerebral microsomes

Characteristics of condensation and overall elongation of very-long-chain fatty-acyl-CoAs in swine cerebral microsomes were studied using radio high-performance liquid chromatography (RHPLC) and gas chromatography-mass spectrometry (GC-MS). The monounsaturated fatty-acyl-CoA depressed both the condensation and overall elongation activities of endogenous substrates and also of exogenous saturated fatty-acyl-CoA. The extent of the decrease of the elongation activity was dependent on the concentration and the chain length of the exogenous fatty-acyl-CoAs. The dependence of the condensation activity of monounsaturated fatty-acyl-CoA on the concentration of malonyl-CoA suggested that the non-Michaelis-Menten type kinetics was dominant for oleoyl-CoA, however, a normal kinetic pattern was obtained for endogenous palmitoyl-CoA and arachidonoyl-CoA with Km = 37 microM to malonyl-CoA. The condensation activity for icosanoyl-CoA (20:0-CoA) was inhibited by icosenoyl-CoA (20:1-CoA) in a non-competitive manner, which suggested that the condensation enzyme, or at least the active center of the enzyme for icosenoyl-CoA, was different from that for icosanoyl-CoA.     

Concomitant decrease in the elongation and condensation activities of very-long chain fatty acyl-CoA in jimpy mouse

Overall elongation and condensation of long-chain and very-long-chain fatty acids have been studied in the brain microsomes of jimpy mice. Both the elongation and condensation activities with stearoyl (18:0)-, oleoyl (18:1)- and arachidoyl (20:0)-CoA were severely diminished in jimpy brain, but the decrease in the activity with the exogenous palmitoyl (16:0)-CoA was less pronounced. The decrease in the elongation and condensation reactions with endogenous palmitic and arachidonic (20:4) acids was not distinct in the mutant. The decrease in the activity of condensation reaction may be responsible for the reduced rate of overall fatty acid elongation.     

Effect of phospholipase A2 and free fatty acids on lipid-protein interactions in long- and very-long-chain fatty acyl-CoA elongation enzyme systems of brain microsomes

The elucidation of the mechanism of phospholipase A2-induced inactivation of the condensation enzyme provided evidence concerning the important role of lipid-enzyme interactions in maintaining the condensation activity in swine cerebral microsomes. A quantitative analysis of fatty acid release by phospholipase A2 from the microsomal membrane revealed that only 5 nmol of free fatty acid per mg microsomal protein was released, including oleic acid and arachidonic acid, by treatment with 0.4 unit of phospholipase A2 per mg microsomal protein for 15 s at 23 degrees C. Under these conditions, the condensation activity for endogenous 16:0-CoA and 20:4-CoA decreased to half and that for exogenous 20:0-CoA decreased to 75%. However, the addition of free fatty acids and lysophospholipids or a mixture of them at 5-10 nmol/mg protein did not change the condensation activity for endogenous 16:0-CoA and 20:4-CoA, or for exogenous 20:0-CoA. These results indicated that phospholipase A2 inhibited the condensation activity by acting directly on phospholipids that are indispensable to maintaining the function of the condensation enzyme. The Arrhenius plot for the condensation of endogenous 16:0-CoA showed a break at around 16 degrees C, whereas no break of the plot was observed for the condensation of 20:0-CoA and 20:4-CoA. The activation energy for the condensation of 16:0-CoA and 20:4-CoA was decreased by the addition of free fatty acids such as oleic acid and stearic acid, with disappearance of the Arrhenius break for 16:0-CoA condensation, whereas the activation energy for the condensation of 20:0-CoA was not changed. These results suggest that the type of lipid-protein interaction in the condensation enzyme for 20:0-CoA is different from that for 16:0-CoA and 20:4-CoA.     

Arachidoyl- and arachidonoyl-CoA elongation mechanism in swine cerebral microsomes

Long-chain saturated and polyunsaturated fatty acyl-CoA elongations were studied in swine cerebral microsomes. The elongation of endogenous palmitoyl-CoA to stearate was highly active in both cerebral and liver microsomes, whereas those of arachidoyl-CoA (20:0-CoA) and endogenous arachidonoyl-CoA (20:4-CoA) were high in cerebral microsomes, but negligible in liver microsomes. The elongation of 22:4 to 24:4 was also observed in cerebral microsomes. Both NADPH and NADH at 500 microM were effective in elongation of 16:0-, 20:0- and 20:4-CoA, whereas NADPH was more effective in elongation of 22:4 to 24:4 than NADH. The incorporation of deuterium atoms to the elongated product was detected by the technique of mass fragmentography when the NADPH-dependent elongations of 20:0-CoA and 20:4-CoA were performed in 2H2O medium upon cerebral microsomes. The number of incorporated deuterium atoms into 22:0 elongated from 20:0-CoA was mainly two, and that into 22:4 elongated from 20:4-CoA was mainly three. These results indicated that part of hydrogens in elongated arachidoyl- and arachidonoyl-CoA were transferred from NADPH.     

Tissue and chain length specificity of the fatty acyl-CoA elongation system in the American cockroach

The elongation of fatty acyl-CoAs, reactions involved in hydrocarbon biosynthesis, was examined in the cockroach, Periplaneta americana. Products were analyzed by radio-HPLC and radio-GLC. The majority of the elongation activity was observed in microsomes prepared from abdominal epidermal tissue. Linoleoyl-CoA (18:2-CoA) was elongated most efficiently followed by stearoyl-CoA (18:0-CoA), linolenoyl-CoA (18:3-CoA; n-3) and oleoyl-CoA (18:1-CoA). The products of 18:2-CoA elongation included all even numbered acyl groups up to 28 carbons, and the products of 18:0-CoA included all even numbered acyl groups to 26 carbons. The 18:3-CoA was elongated only to 20 and 22 carbons. Radioactivity from both 18:2-CoA (5.4%) and 18:0-CoA (1.2%) was recovered in the hydrocarbon fraction. Analysis of this hydrocarbon fraction showed that the radio-activity from 18:2-CoA was present in (Z,Z)-6,9-heptacosadiene and that the radioactivity from 18:0-CoA was present in n-pentacosane. These data demonstrate for the first time in an in vitro insect system that the fatty acid elongation reactions are coupled with the conversion of the elongated product to hydrocarbon. Thus, each of the expected intermediates in the conversion of 18:0 and 18:2 to 25 and 27 carbon hydrocarbons, respectively, was observed, and the results demonstrate high tissue, substrate, and product specificity.     

Characterization of fatty acid elongation system in porcine neutrophil microsomes

Microsomes purified from porcine neutrophils containing the fatty acid chain-elongation system for long- and very-long-chain fatty acyl-CoAs, and several enzymatic characters for the elongation of palmitoyl-CoA (16:0-CoA) and arachidoyl-CoA (20:0-CoA) were examined. The heat-inactivation profile for the elongation of 16:0-CoA was different from that of 20:0-CoA, suggesting the presence of different enzyme systems for palmitoyl-CoA and arachidoyl-CoA. Contrary to the elongation system of brain microsomes, the successive synthesis of lignoceric acid (24:0) from 20:0-CoA at 60 microM was not prominent under normal conditions in the neutrophil microsomes. The synthesis of behenic acid (22:0) was slightly inhibited by 0.5 mM N-ethylmaleimide (NEM) present in the assay mixture, whereas the pre-treatment of microsomes with 0.5 mM NEM largely inhibited the synthesis of 22:0 from 20:0-CoA. The synthesis of 24:0, however, was enhanced by 0.5 mM NEM in the elongation of 20:0-CoA and the rate of 24:0 synthesis became dominant over the synthesis of 22:0. These results suggested that the elongation enzyme for very-long-chain fatty acyl-CoA, especially for 20:0-CoA elongation to 22:0 in the neutrophil microsomes contained NEM-sensitive sulfhydryl groups in the active center and the mechanism for the synthesis of 24:0 through successive elongation from 20:0-CoA was different from that of 22:0, as the former was enhanced by NEM whereas the latter was strongly inhibited.     

Effect of fatty-acyl-CoAs on the elongation of saturated fatty acid in porcine aorta microsomes

The microsomal elongation system from porcine aorta for longchain fatty-acyl-CoAs was investigated. Palmitoleoyl-CoA (16:1-CoA), oleoyl-CoA (18:1-CoA), and eicosenoyl-CoA (20:1-CoA) remarkably depressed the elongation activity for 16:0-CoA in aorta microsomes by 44.8, 52.4, and 43.7% of the control activity, respectively. Saturated and polyunsaturated fatty-acyl-CoAs had little effect on the 16:0-CoA elongation activity. These results indicate that monounsaturated long-chain fatty acyl-CoAs can regulate the synthesis of saturated fatty acids in the vessel walls.     

Chain elongation in the formation of polyunsaturated fatty acids by brain: Some properties of the microsomal system

Chain elongation of polyunsaturated acids has been investigated using microsomes from developing rat brain. With 18:3(n ? 6) in 0.05% detergent as an acceptor and [2-¹⁴C]malonyl-coenzyme A (CoA) as a two-carbon donor, incorporation of radioactivity into 20:3 was optimal (and incorporation into other acyl chains was minimal) in the presence of 100 μm substrate, 200 μmp-bromophenacylbromide and 10 mm KCN. Up to 30% of the labeled products were incorporated into phospholipids and triacylglycerol. Maximal microsomal elongation activity was observed at 3–4 weeks of age. Several other fatty acid or acyl-CoA acceptors tested in this system were elongated at slower rates compared to 18:3(n ? 6) [e.g., 16:0-CoA, 75%; 20:4(n ? 6), 57%; 18:3(n ? 3), 13%; 18:2(n ?6), 10%; 20:3(n ? 6), 6%]. The rate of elongation of chemically synthesized 18:3-CoA was only 50% of the detergent-suspended acid and was optimal at 6 μm substrate; inhibition above 6 μm 18:3-CoA was reduced by bovine serum albumin, but incorporation of label into palmitate was greatly stimulated. CoA markedly inhibited elongation of 18:3(n ? 6) or 18:3-CoA; N-ethylmaleimide at equimolar amounts reversed this CoA inhibition but did not alter the inhibition caused by concentrations of 18:3-CoA above 6 μm. ATP was absolutely required for elongation of either the free acid or the acyl-CoA derivative, whereas exogenous MgCl₂ had little effect.     

Solubilization of C18-Co A and C20-CoA elongases from Allium porrum L. epidermal cell microsomes

The effects of n-octyl-β-D-glucopyranoside, Triton X-100 and deoxycholate on acyl-CoA elongation by Allium porrum L. epidermal cell microsomes showed that the Triton X-100 specifically stimulated the synthesis of C22–C26 acids using C18-CoA as primer, whereas the fatty acid elongation products of C20-CoA remained essentially unchanged. n-Octyl-β-D-glucopyranoside increased the C20 and C22 fatty acid syntheses to the same extent and deoxycholate inhibited C18-CoA and C20-CoA elongation. The presence of two different elongation systems, as suggested by these results, has been demonstrated. After solubilization by Triton X-100, the C18-CoA and C20-CoA elongases were separated by sucrose density centrifugation. The fractions corresponding to sucrose concentrations of 0.51 and 0.62 M presented the maximal activities for C18-CoA and C20-CoA elongases, respectively. In addition, by gel filtration on a Sephacryl S-300 column, the C20-CoA and the C18-CoA elongases have estimated apparent molecular masses under detergent conditions of 600 and 350 kDa, respectively.     

Oleoyl-CoA is not an immediate substrate for fatty acid elongation in developing seeds of Brassica napus

The substrate specificity of fatty acid elongase was studied using an oil body fraction from developing seeds of Brassica napus. ATP was essential for high rates of elongase activity, but there was no apparent requirement for oleoyl-CoA, oleic acid (18:1) or CoA. Furthermore, ¹⁴C from 18:1-CoA was incorporated into eicosenoic (20:1) and erucic (22:1) acids at a much slower rate than ¹⁴C from malonyl-CoA. Incubation of [¹⁴C]18:1-CoA with the oil body fraction resulted in a rapid loss of [¹⁴C]18:1-CoA into several lipid fractions whether in the absence or presence of ATP, but the loss of 18:1-CoA had a comparatively small effect on the overall rate of elongation. Acyl-CoAs were derivatized to their respective acylbutylamide and analyzed by gas chromatography-mass spectrometry. This analysis of acyl-CoAs demonstrated that there was no detectable 20:1-CoA or 22:1-CoA at 0 min incubation, while newly synthesized 20:1-CoA and 22:1-CoA were present at 10 min. Analysis of the %¹⁴C of the substrates and products of the elongation reaction revealed that the endogenous pool of 18:1-CoA is quite small in elongase preparations. In addition, [¹⁴C]18:1-CoA added to the incubation, although incorporated into lipids, was not significantly diluted by turnover or new synthesis. In contrast, the %¹⁴C of the 20:1-CoA was two- to threefold less than that of the 18:1-CoA. Taken together, these results indicate that the [¹⁴C]18:1 from the [¹⁴C]18:1-CoA was diluted in an intermediate 18:1 pool and that the 18:1-CoA was not the major donor of the acyl group to the elongase reaction.     

Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi.

Bioluminescent bacteria require myristic acid (C14:0) to produce the myristaldehyde substrate of the light-emitting luciferase reaction. Since both endogenous and exogenous C14:0 can be used for this purpose, the metabolism of exogenous fatty acids by luminescent bacteria has been investigated. Both Vibrio harveyi and Vibrio fischeri incorporated label from [1-14C]myristic acid (C14:0) into phospholipid acyl chains as well as into CO2. In contrast, Photobacterium phosphoreum did not exhibit phospholipid acylation or beta-oxidation using exogenous fatty acids. Unlike Escherichia coli, the two Vibrio species can directly elongate fatty acids such as octanoic (C8:0), lauric (C12:0), and myristic acid, as demonstrated by radio-gas liquid chromatography. The induction of bioluminescence in late exponential growth had little effect on the ability of V. harveyi to elongate fatty acids, but it did increase the amount of C14:0 relative to C16:0 labeled from [14C]C8:0. This was not observed in a dark mutant of V. harveyi that is incapable of supplying endogenous C14:0 for luminescence. Cerulenin preferentially decreased the labeling of C16:0 and of unsaturated fatty acids from all 14C-labeled fatty acid precursors as well as from [14C]acetate, suggesting that common mechanisms may be involved in elongation of fatty acids from endogenous and exogenous sources. Fatty acylation of the luminescence-related synthetase and reductase enzymes responsible for aldehyde synthesis exhibited a chain-length preference for C14:0, which also was indicated by reverse-phase thin-layer chromatography of the acyl groups attached to these enzymes. The ability of V. harveyi to activate and elongate exogenous fatty acids may be related to an adaptive requirement to metabolize intracellular C14:0 generated by the luciferase reaction during luminescence development.     

Condensation activity for polyunsaturated fatty acids with malonyl-CoA in rat brain microsomes. Characteristics and developmental change.

Condensation activities for gamma-linolenic acid (18:3(n-6)), octadecatetraenoic acid (18:4(n-3)) and eicosapentaenoic acid (20:5(n-3)) with malonyl-CoA were measured and compared with the condensation activities for 16:0-CoA, 18:1-CoA, 18:2(n-6)-CoA and 18:3(n-3)-CoA in rat brain microsomes of various ages. The age-dependence of condensation activities for 18:3(n-6), 18:4(n-3) and 20:5(n-3) showed a maximum at 1- to 2-month-old and were still higher at 3-month-old 2- to 3-fold than the activities in microsomes of pups. Conversely, the age-dependence of condensation activity for 16:0-CoA showed a peak around 1 month-old, but decreased at 3-month-old to the level of the activities in pups. The condensation activity for 20:5(n-3) was inhibited by 18:3(n-6) or 18:4(n-3) and the inhibition was not competitive. The condensation of 18:3(n-6) was also inhibited by 18:4(n-3) in the same manner. A physiological implication of the inhibition system at the substrate level was discussed.     

Characteristics of Synthesis of Very-Long-Chain Saturated and Tetraenoic Fatty Acids in Swine Cerebral Microsomes

The elongation of arachidoyl-CoA (20:0-CoA) yielded 22:0 and 24:0 concomitantly, whereas the elongation of behenoyl-CoA (22:0-CoA) yielded only a negligible amount of 24:0 in adult swine cerebral microsomes. The dependence on time, pH, and the substrate concentrations were examined for the synthesis of 22:0 and 24:0 from 20:0-CoA. A microcomputer-aided simulation study suggested that there were two parallel pathways in the elongation of 20:0-CoA to 22:0 and 24:0. The elongation of 22:0-CoA could not be observed in adult swine cerebral microsomes; however, it was observed clearly in newborn swine and rat brain microsomes. A dilution experiment with the addition of cold 22:0-CoA in the reaction of elongation of 20:0-CoA confirmed the above suggestion that no intermediate 22:0 appeared during the synthesis of 24:0 from 20:0-CoA. The elongation of endogenous 20:4-CoA to 22:4 and 24:4 was examined in newborn swine cerebral microsomes, and the presence of two parallel pathways in the elongation of 20:4-CoA to 22:4 and 24:4 similar to those involved in the elongation of 20:0-CoA to 22:0 and 24:0 was suggested.     

Regulation of sex pheromone biosynthesis in the housefly, Musca domestica: relative contribution of the elongation and reductive steps.

The regulation of production of the sex pheromone (Z)-9-tricosene (Z9-23:Hy) in the housefly, Musca domestica, was studied by examining the chain length specificity of the fatty acyl-CoA elongation reactions and the reductive conversion of fatty acyl-CoAs to alkenes in 1- and 4-day-old male and female houseflies. Microsomal preparations from 4-day-old female insects produced as the predominant alkene Z9-23:Hy when incubated with malonyl-CoA, NADPH, and [9,10-3H2]oleoyl-CoA (18:1-CoA), whereas microsomal preparations from 4-day-old male insects produced predominantly (Z)-9-heptacosene (Z9-27:Hy). These are the major alkenes produced in vivo by Day 4 females and males, respectively. Microsomes prepared from both Day 1 males and Day 1 females produced Z9-27:Hy as the major alkene from labeled 18:1-CoA. This is the major alkene produced in vivo by both sexes at Day 1. An examination of the chain length specificity of the elongation reactions showed that microsomes prepared from Day 4 male insects readily elongated both 18:1-CoA and 15-[15,16-3H2]tetracosenoyl-CoA (24:1-CoA) to 28-carbon moieties, whereas microsomes from Day 4 female insects did not efficiently elongate either substrate beyond 24 carbons. With high substrate concentrations, microsomes prepared from male insects converted 24:1-CoA to Z9-23:Hy more efficiently than did those from females, whereas under lower and presumably more physiological substrate concentrations, microsomes from females had slightly higher activity than did those from males. Taken together, these data show that the regulation of the chain length of the alkenes, and thus sex pheromone production, in the housefly resides predominantly in the elongation reactions and not in the step which converts the fatty acyl-CoA to hydrocarbon.     

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Synthesis in vitro of very long chain fatty acids in Vibrio sp. strain ABE-1

The activity of fatty acid synthetase (FAS) from Vibrio sp. strain ABE-1 required the presence of acyl carrier protein and was completely inhibited by thiolactomycin, an inhibitor specific for a type II FAS. These observations indicate that this enzyme is a type II FAS. Analysis by gas-liquid chromotography of the reaction products synthesized in vitro from [2-¹⁴C]malonyl-CoA by the partially purified FAS revealed, in addition to 16-and 18-carbon fatty acids which are normal constituents of this bacterium, the presence of fatty acids with very long chains. These fatty acids were identified as saturated and mono-unsaturated fatty acids with 20 up to as many as 30 carbon atoms. The longest fatty acids normally found in this bacterium contain 18-carbon atoms. These results suggest that the FAS from Vibrio sp. strain ABE-1 has potentially the ability to synthesize fatty acids with very long chains.Abbreviations ACP acyl carrier protein - FAME fatty acid methyl ester - FAS fatty acid synthetase - FID flame ionization detection - GLC gas-liquid chromatography - TLC thin-layer chromatography - In designations of fatty acids, such as 16:0, 16:1, etc the colon separates the number that denotes the number of carbon atoms and the number that denotes the number of double bonds, respectively, in the molecule - 16:0-CoA CoA ester of 16:0     

Site of inhibition of rat liver microsomal fatty acid chain elongation system by dec-2-ynoyl coenzyme A. Possible mechanism of inhibition

The present study examines the effect of the acetylenic thioester dec-2-ynoyl-CoA (delta 2 10 identical to 1-CoA) on the microsomal fatty acid chain elongation pathway in rat liver. When the individual reactions of the elongation system were measured in the presence of delta 2 10 identical to 1-CoA, the trans-2-enoyl-CoA reductase activity was markedly inhibited (Ki = 2.5 microM), whereas the activities of the condensing enzyme, the beta-ketoacyl-CoA reductase, and the beta-hydroxyacyl-CoA dehydrase were not affected. The absence of inhibition of total microsomal fatty acid elongation was attributed to the significant accumulation of the intermediates, beta-hydroxyacyl-CoA and trans-2-enoyl-CoA, without formation of the saturated elongated product, indicating that the trans-2-enoyl-CoA reductase-catalyzed reaction was the only site affected by the inhibitor. The nature of the inhibition was noncompetitive. In contrast to the delta 2 10 identical to 1-CoA, delta 3 10 identical to 1-CoA did not inhibit trans-2-enoyl-CoA reductase activity, suggesting that the mode of inhibition was not via formation of the 2,3-allene derivative. Based on the observation (a) that p-chloromercuribenzoate markedly inhibits reductase activity, (b) that dithiothreitol protects the enzyme against inactivation by delta 2 10 identical to 1-CoA, (c) of the spectral manifestation of the interaction between thiol reagents and delta 2 10 identical to 1-CoA depicting an absorbance peak similar to that of the beta-ketoacyl thioester-Mg2+ enolate complex, (d) of a similar absorbance spectrum formed by the interaction between delta 2 10 identical to 1-CoA and liver microsomes, and (e) of the absence of formation of a similar spectrum by delta 3 10 identical to 1-CoA, trans-2-10:1-CoA, or delta 2 10 identical to 1 free acid with liver microsomes, we propose that delta 2 10 identical to 1-CoA inactivates trans-2-enoyl-CoA reductase by covalently binding to a critical sulfhydryl group at or in close proximity to the active site of the enzyme.     

Identification of Amino Acids Conferring Chain Length Substrate Specificities on Fatty Alcohol-forming Reductases FAR5 and FAR8 from Arabidopsis thaliana

Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.     

Aliphatic analogues of nucleotides: synthesis and affinity towards nucleases

DL-1-(2,3-Dihydroxypropyl)thymine was prepared by Hilbert-Johnson reaction of 2,4-dinethoxy-5-methylpyrimidine with allyl bromide followed by the osmium tetroxide catalyzed hydroxylation of the l-allyl-4-methoxy-5-methylpyrimidin-2-one obtained as an intermediate. The D-glycero enantiomer, R-1-(2,3-dihydroxypropyl)thymine and the corresponding 1-substituted uracil derivative were prepared from 3-O-p-toluenesulfonyl-1, 2-O-isopropylidene-D-glycerine and sodium salt of 4-methoxy-5-methylpyrimidin-2-one or 4-methoxypyrimidin-2-one followed by treatment with hydrogen chloride in ethanol. The phosphorylation of the above 2,3-dihydroxypropyl derivatives with phosphoryl chloride in triethyl phosphate afforded the corresponding 3-phosphates which were transformed into the 2′,3′-cyclic phosphates by the condensation with N,N′-dicyclohexylcarbodiimide. The latter compounds of the D-glycero configuration are split by some microbial RNases to the 3-phosphates.