Relationship between translocation of long-chain acyl-CoA hydrolase,phosphatidate phosphohydrolase and CTP:Phosphocholine cytidylyltransferase and the synthesis of triglycerides and phosphatidylcholine in rat liver

Translocation of long-chain acyl-coenzyme A hydrolase from the microsomal fraction to the cytosolic fraction was promoted in cell-free extracts of rat liver by palmitic acid, oleic acid, tetradecylthioacetic acid, and tetradecylthiopropionic acid, and by their CoA esters. The CoA esters were more effective than the non-esterified acids in the translocation of the enzyme. Treatment of normolipidemic rats with sulfur-substituted non-β-oxidizable fatty acid analogues resulted in a transitory increase in hepatic concentration of long-chain acyl-CoA. Longer feeding times almost normalized the hepatic long-chain acyl-CoA content. Microsomal long-chain acyl-CoA hydrolase activity was inhibited, whereas the activity of the cytosolic form was stimulated. The rise in enzyme activity coincided with a reduction in liver content of triglyceride and an increase in hepatic phospholipid content. The results suggest that the activity of long-chain acyl-CoA hydrolase in the cytosol may control the amount of acyl-CoA thioesters in the liver. Esterified and non-esterified fatty acids causedin vitro translocation of phosphatidate phosphohydrolase and cytidine 5′-triphosphate (CTP):phosphocholine cytidylyltransferase from the cytosolic fraction to the microsomal fraction. However, the translocation of these two enzyme systems was not obtainedin vivo. The activity of phosphatidate phosphohydrolase decreased in microsomal and cytosolic fractions while the activity of cytidylyltransferase in these fractions increased. The activities of soluble phosphatidate phosphohydrolase and long-chain acyl-CoA hydrolase appeared to be inversely correlated. The results imply that in cytoplasm, long-chain acyl-CoA hydrolase may compete with the biosynthetic enzymes for the acyl-CoA substrate, thus influencing the rate of lipid synthesis. The reduced hepatic triglyceride content observed in tetradecylthioacetic acid-treated rats is probably due to reduced triglyceride synthesis, which is mediated by an inhibition of phosphatidate phosphohydrolase accompanied with translocation and stimulation of long-chain acyl-CoA hydrolase. Development of fatty liver as an effect of tetradecylthiopropionic acid is probably due to accelerated triglyceride biosynthesis, which is mediated by a stimulation of phosphatidate phosphohydrolase and a decrease in cytosolic palmitoyl-CoA hydrolase activity.     

Iodothyronamines are Oxidatively Deaminated to Iodothyroacetic Acids in vivo

3‐Iodothyronamine (T₁AM) and 3,3′,5‐triiodothyroacetic acid (Triac) are bioactive metabolites of the hormone thyroxine (T₄). In the present study, the ability of T₁AM and 3,3′,5‐triiodothyronamine (T₃AM) to be metabolized to 3‐iodothyroacetic acid (TA₁) and Triac, respectively, was investigated. Both T₁AM and T₃AM were converted to their respective iodinated thyroacetic acid analogues in both cell and tissue extracts. This conversion could be significantly inhibited with the monamine oxidase (MAO) and semicarbazide‐sensitive amine oxidase (SSAO) inhibitor iproniazid. TA₁ was found to be present in trace quantities in human serum and in substantial levels in serum from T₁AM‐treated rats. These results demonstrate that iodothyronamines are substrates for amine oxidases and that this metabolism may be the source of the corresponding endogenous arylacetic acid products Triac and TA₁.     

4‐Hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐one Synthesis by a Type III Polyketide Synthase from Rhodospirillum centenum

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl‐CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C_10–14 fatty acyl‐CoA unit, one malonyl‐CoA unit and two methylmalonyl‐CoA units. We identified these products as 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl‐CoA as the extender substrate. In addition, in vitro reactions with ¹³C‐labeled malonyl‐CoA revealed that RpsA produced tetraketide 6‐alkyl‐4‐hydroxy‐1,5‐dimethyl‐2‐oxocyclohexa‐3,5‐diene‐1‐carboxylic acids from C_14–20 fatty acyl‐CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.     

Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega‐3 fatty acids that provide important human health benefits. Canola (Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl‐CoA substrates in the presence of malonyl‐CoA. To distinguish between enzymes that desaturate CoA‐ and phosphatidylcholine‐linked fatty acid substrates, we developed a novel in vitro method. We show that a delta‐12 desaturase from Phytophthora sojae, an omega‐3 desaturase from Phytophthora infestans and a delta‐4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine‐linked acyl substrates with comparatively low use of acyl‐CoA substrates. To further validate our method, a delta‐9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl‐CoA as substrate, but could not use phosphatidylcholine‐linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants.     

Structural Basis of Cyclic 1,3-Diene Forming Acyl-Coenzyme A Dehydrogenases

The biologically important, FAD-containing acyl-coenzyme A (CoA) dehydrogenases (ACAD) usually catalyze the anti-1,2-elimination of a proton and a hydride of aliphatic CoA thioesters. Here, we report on the structure and function of an ACAD from anaerobic bacteria catalyzing the unprecedented 1,4-elimination at C3 and C6 of cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA) to cyclohex-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) and at C3 and C4 of the latter to benzoyl-CoA. Based on high-resolution Ch1CoA dehydrogenase crystal structures, the unorthodox reactivity is explained by the presence of a catalytic aspartate base (D91) at C3, and by eliminating the catalytic glutamate base at C1. Moreover, C6 of Ch1CoA and C4 of Ch1,5CoA are positioned towards FAD-N5 to favor the biologically relevant C3,C6- over the C3,C4-dehydrogenation activity. The C1,C2-dehydrogenation activity was regained by structure-inspired amino acid exchanges. The results provide the structural rationale for the extended catalytic repertoire of ACADs and offer previously unknown biocatalytic options for the synthesis of cyclic 1,3-diene building blocks.     

Identification and Biosynthesis of New Acyloins from the Thermophilic Bacterium Thermosporothrix hazakensis SK20‐1T

Two new acyloin compounds were isolated from the thermophilic bacterium Thermosporothrix hazakensis SK20‐1^T. Genome sequencing of the bacterium and biochemical studies identified the thiamine diphosphate (TPP)‐dependent enzyme Thzk0150, which is involved in the formation of acyloin. Through extensive analysis of the Thzk0150‐catalyzed reaction products, we propose a putative reaction mechanism involving two substrates: 4‐methyl‐2‐oxovalerate as an acyl donor and phenyl pyruvate as an acyl acceptor.     

Studies on culture condition of new marine bacterium Zooshikella sp. SY01

New marine bacterium Zooshikella sp. SY01, producer of prodigiosin, was isolated from the seawaters of Sanya Bay. The culture conditions of this bacterium were investigated. Zooshikella sp. SY01 was cultured in 2216E media which contained tryptophan, histidine, lactonic acid, camphor, limonene, casein, diphenyl guanidine, coumarin and 1,3-dinitrobenzene, respectively. After 5 days cultivation, the extracts of different culture broths were detected by direct infusion mass spectroscopy using positive ESI mode. As the results, tryptophan, histidine and casein didn’t show any observable influences on the biosynthesis of prodigiosin. Lactonic acid, camphor, limonene, diphenyl guanidine, coumarin could inhibit the bacterium growth and prodigiosin biosynthesis to a certain extent, slower the culture broth to turn red. However, 1,3-dinitrobenzene inhibited the bacteria to produce prodigiosin completely. MS data suggested that various metabolites with chemodiversity were produced in different culture media. In particular, a series of high-molecular-weight compounds with high relative abundances were observed in the medium containing limonene. To further optimize the culture condition, more new prodigiosin analogues and lead compounds can be obtained and the goal of “one strain-many compounds” can be achieved. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2007, 46(6): 55–58 [译自: 中山大学学报(自然科学版)]     

Evidence for the mitochondrial biosynthesis of 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid in the yeast Dipodascopsis uninucleata

The biosynthesis of 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (3R-HETE) from arachidonic acid (20∶4n−6) by the hyphal-forming yeast, Dipodascopsis uninucleata, in cell-free enzyme extracts required CoASH, ATP, NAD⁺ and Mg²⁺; 3R-HETE was present as the CoA derivative in enzyme extracts and its biosynthesis was associated with mitochondria. Its synthesis was high from arachidonoyl-CoA (15% conversion of the substrate; 22 nmol mg protein⁻¹·h), but significantly higher from trans-2-arachidonoyl-CoA (53 nmol mg protein⁻¹·min). Aspirin, an inhibitor of prostaglandin endoperoxide synthase synthase (cyclooxygenase), did not significantly inhibit 3R-HETE biosynthesis in enzyme extracts, as opposed to antimycin A (46% inhibition). The chirality of 3-HETE was 95% R and 5% S. 3R-HETE has the same chirality as the products of peroxisomal enoyl-CoA hydratases of Neurospora crassa and Saccharomyces cerevisiae; the difference appears to be that in D. uninucleata the R-enantiomers are synthesized in mitochondria. Exogenously supplied eicosapentaenoic acid was converted to 3-hydroxy 5Z,11Z,14Z,17Z-eicosapentaenoic acid by cell-free enzyme extracts though there was no requirement for a 5Z,8Z-diene structure for the biosynthesis of 3-hydroxylated fatty acids as 3-hydroxy-8Z,11Z,14Z, and 3-hydroxy-11Z,14Z,17Z-eicosatrienoic acids were synthesized from the corresponding fatty acids. We found no evidence for the synthesis of the prostaglandins F_2α and F₂.     

Comparative effects of docosa-4,7,10,13,16-pentaenoic acid and docosa-4,7,10,13,16,19-hexaenoic acid on the desaturation of linoleic acid and α-linolenic acid

Varying concentrations of free docosa-4,7,10,13,16-pentaenoic acid or its CoA ester were incubated with a given variable concentration of 1-¹⁴C-linoleate or 1-¹⁴C-α-linolenate as either the free fatty acid or the CoA ester, microsomal enzymes, and the appropriate cofactors for fatty acid desaturation. The results obtained were compared to the effects of docosa-4,7,10,13,16,19-hexaenoyl CoA when incubated in a similar manner in the presence of the labeled substrates. Both feedback and crossed inhibition effects were observed; these inhibition effects may play a role in the regulation of polyunsaturated fatty acid biosynthesis.     

Heterologous Expression of an Unusual Ketosynthase,SxtA, Leads to Production of Saxitoxin Intermediates in Escherichia coli

Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage-gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4-amino-3-oxo-guanidinoheptane (ethyl ketone) by an unusual polyketide synthase-like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity in vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC-ESI-HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity in vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled-precursor feeding with [guanidino-¹⁵N₂] arginine and [1,2-¹³C₂] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.     

Pathways of acetyl CoA production for lipogenesis from acetoacetate, β-hydroxybutyrate,pyruvate and glucose in neonatal rat lung

The rate of fatty acid synthesis from acetoacetate (AcAc) is 2–3 times greater than from glucose in developing rat lung. To determine the reason for this difference, we investigated the pathways of lipogenesis from [3-¹⁴C] AcAc, [3-¹⁴C]β-hydroxybutyrate (βOHB), [U-¹⁴C] glucose or [2-¹⁴C] pyruvate in minced lung tissue of 3- to 4-day-old rats. The addition of (−)hydroxycitrate, an inhibitor of ATP-citrate lyase, inhibited fatty acid synthesis from glucose, pyruvate, and βOHB by 88%, 70% and 60%, respectively, but had no effect on that from AcAc. Benzene 1,2,3-tricarboxylate, an inhibitor of tricarboxylate translocase, inhibited fatty acid synthesis from all substrates by at least 50%. Incubation with aminooxyacetate, an inhibitor of aspartate aminotransferase, has no effect on lipid synthesis from glucose, pyruvate or AcAc, but increased lipid synthesis from βOHB. Results indicate that for lipid synthesis in the neonatal lung, acetyl CoA from AcAc is derived predominantly from a cytoplasmic pathway involving AcAcCoA synthetase and AcAcCoA thiolase, whereas citrate is the major route of acetyl group transfer from glucose. Lipogenesis from βOHB involves both the cytoplasmic and citrate pathways.     

Synthesis and Enzymic Conversion of Isobutanoyl-carba(dethia)-Coenzyme A

Acyl coenzyme A analogues in which the sulphur is replaced by methylene are stable to hydrolysis and can be used to investigate enzymatic reaction mechanisms. With this idea in mind we synthesized isobutanoyl-carba(dethia)-coenzyme A (isobutanoyl-CH₂CoA) and monitored its reaction in enzymatic systems. A cell-free extract of Pseudomonas putida (ATCC 21244) complemented with FAD, phenazine methosulphate, and DCIP catalyzed the conversion of isobutanoyl-CH₂CoA into methacryloyl-CH₂CoA and β-hydroxyisobutanoyl-CH₂CoA. The latter could not be further oxidized to the aldehyde and carboxyl level, indicating that these oxidations normally occur after hydrolysis of the CoA ester.     

Uptake and metabolism of fatty acids by Soybean suspension cells

Soybean suspension cultures very rapidly take up C₁₆ and C₁₈ fatty acids by a nonspecific, nonenzymic binding of exogeneously added fatty acids to cell walls and by a subsequent transfer into the cell where they are rapidly incorporated into triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines.¹⁴C-Palmitic and¹⁴C-stearic acids follow this sequence but are not desaturated, wherease¹⁴C-oleic and¹⁴C-linoleic acids are transferred more rapidly than the saturated fatty acids and are then further modified. All the data fit a sequence of events by which free oleic acid is first activated to a CoA thioester, and then desaturated to linoleyl-CoA; both thioesters are then transferred to triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine.     

Biosynthesis of Phenylnannolone A,a Multidrug Resistance Reversal Agent from the Halotolerant Myxobacterium Nannocystis pusilla B150

The myxobacterial strain Nannocystis pusilla B150 synthesizes the structurally new polyketides phenylnannolone A–C. Apart from some common volatiles and siderophores, these are the first natural products from the genus Nannocystis. Phenylnannolone A shows inhibitory activity towards the ABCB1 gene product P‐glycoprotein and reverses daunorubicin resistance in cancer cells. To decipher the biochemical reactions leading to the formation of phenylnannolone A, the putative biosynthetic genes were identified (phn1, phn2). Phn2 is a polyketide synthase (PKS) with an NRPS‐like loading module, and its domain order is consistent with the phenylnannolone A structure. The functionality and substrate selectivity of the loading module were determined by means of a γ‐¹⁸O₄‐ATP pyrophosphate exchange and a phosphopantetheine ejection assay. A specific activation of cinnamic acid by the AMP‐ligase was detected. Phn1 is a putative butyryl‐CoA carboxylase (BCC), providing ethylmalonyl‐CoA for the formation of the ethyl‐substituted part of phenylnannolone A. Phn1 is the first BCC found in biosynthetic genes for an ethyl‐substituted natural compound. Biosynthesis of phenylnannolone A, putatively encoded by phn1 and phn2, thus utilizes the first biosynthetic machinery in which both a BCC and a PKS are involved.     

Effects of ultrasonic pretreatments on thermodynamic properties,water state,color kinetics,and free amino acid composition in microwave vacuum dried lotus seeds

Abstract

Ultrasonic pretreatments were applied to lotus seeds at acoustic energy densities of 0.29, 0.40, and 0.51?W mL^?1 for 10?min. After pretreatments, lotus seeds were subjected to microwave vacuum drying (MVD). Parameters of glass transition temperature (T_g), gelatinization temperature (T_p), water state, color kinetics, and free amino acid content of microwave vacuum dried lotus seeds were determined. With increasing acoustic energy density, MVD elevated the T_g values appreciably by decreasing the content of cytoplasmic bulk water in lotus seeds tissues. The T_p had a positive relationship with the relaxation times of cytoplasmic bulk water (T₂₂), while T_g had a negative relationship with T₂₂. Color kinetics were analyzed by the divisional method during MVD due to different browning reactions, which failed to appear with ultrasonic pretreatment. Free amino acid content ranged from 517.65 to 666.13?mg/100?g dry weight at 0.51?W mL^?1.     

A Tandem Enzymatic sp2‐C‐Methylation Process: Coupling in Situ S‐Adenosyl‐l‐Methionine Formation with Methyl Transfer

A one‐pot, two‐step biocatalytic platform for the regiospecfic C‐methylation and C‐ethylation of aromatic substrates is described. The tandem process utilises SalL (Salinospora tropica) for in situ synthesis of S‐adenosyl‐l ‐methionine (SAM), followed by alkylation of aromatic substrates by the C‐methyltransferase NovO (Streptomyces spheroides). The application of this methodology is demonstrated for the regiospecific labelling of aromatic substrates by the transfer of methyl, ethyl and isotopically labelled ¹³CH_3, ¹³CD₃ and CD₃ groups from their corresponding SAM analogues formed in situ.     

Rapid Screening,Identification, Separation,and Purification of Four Bioactive Compounds from Swertia mussotii Franch

Gentiopicroside, mangiferin, sweroside, and isoorientin, the bioactive constituents of Swertia mussotii Franch, have various pharmacological effects, and are used in particular for treating liver disorders. However, efficient methods for their separation are not currently available. In this study, these bioactive compounds were detected in S. mussotii extracts using high-performance liquid chromatography coupled with mass spectrometry, and separated using a combination of high-speed counter-current chromatography and Sephadex LH-20 column chromatography. Their structures were determined by using ¹H and ¹³C nuclear magnetic resonance spectroscopies. The results show that this method is effective for the separation and purification of bioactive compounds from S. mussotii.     

Diacylglycerol acyltransferases from Vernonia and Stokesia prefer substrates with vernolic acid

Genetic engineering of common oil crops for industrially valuable epoxy FA production by expressing epoxygenase genes alone had limited success. Identifying other key genes responsible for the selective incorporation of epoxy FA into seed oil in natural high accumulators appears to be an important next step. We investigated the substrate preferences of acyl CoA: diacylglycerol acyltransferases (DGAT) of two natural high accumulators of vernolic acid, Vernonia galamensis and Stokesia laevis, as compared with a common oilseed crop soybean. Developing seed microsomes were fed with either [¹⁴C]oleoyl CoA or [¹⁴C]vernoloyl CoA in combinations with no exogenous DAG or with 1,2-dioleoyl-sn-glycerol, 1-palmitoyl-2-vernoloyl-sn-glycerol, 1,2-divernoloyl-sn-glycerol, 1,2-dioleoyl-rac-glycerol, or 1,2-divernoloyl-rac-glycerol to determine their relative incorporation into TAG. The results showed that in using sn-1,2-DAG, the highest DGAT activity was from the substrate combination of vernoloyl CoA with 1,2-divernoloyl-sn-glycerol, and the lowest was from vernoloyl CoA or oleoyl CoA with 1,2-dioleoyl-sn-glycerol in both V. galamensis and S. laevis. Soybean DGAT was more active with oleoyl CoA than vernoloyl CoA, and more active with 1,2-dioleoyl-sn-glycerol when oleoyl CoA was fed. DGAT assays without exogenous DAG, or with exogenous sn-1,2-DAG fed individually or simultaneously showed consistent results. In combinations with either oleoyl CoA or vernoloyl CoA, DGAT had much higher activity with rac-1,2-DAG than with their corresponding sn-1,2-DAG, and the substrate selectivity was diminished when rac-1,2-DAG were used instead of sn-1,2-DAG. These studies suggest that DGAT action might be an important step for selective incorporation of vernolic acid into TAG in V. galamensis and S. laevis.     

Intermediates and products formed during fatty acid α-oxidation in cucumber (Cucumis sativus)

Fatty acid α-oxidation is an essential metabolic pathway both in plants and in mammals which is still not completely understood. We previously described and purified an α-oxidation enzyme in cucumber which has been used in the present investigation of the α-oxidation reaction mechanism. Free fatty acids, and not the CoA thioesters, were found to undergo α-oxidation in cucumber. 2-Hydroxy- and 2-oxopalmitic acids were identified as palmitic acid α-oxidation intermediates by high-performance liquid chromatography and gas chromatography—mass spectrometry analysis in cucumber subcellular 150,000×g _max pellets obtained by differential centrifugation. Incubation of purified α-oxidation enzyme with [1-¹⁴C]palmitic acid resulted in the formation of both the above-described intermediates and the C _n−1 product, pentadecanal, and ¹⁴CO₂. Besides ¹⁴CO₂, ¹⁴C-formate was identified as an α-oxidation product from [1-¹⁴C]palmitic acid in cucumber subcellular fractions. Fe²⁺ stimulated the ¹⁴CO₂ and ¹⁴C-formate production, and the addition of ascorbate and 2-oxoglutarate together with Fe²⁺ resulted in optimal α-oxidation activities, suggesting a dioxygenase reaction mechanism, as previously shown in mammals. NADPH and, to a lesser extent, NADH stimulated the total ¹⁴C-formate plus ¹⁴CO₂ production but had only slight or no effects on ¹⁴CO₂ production. H₂O₂ showed concentration-dependent inhibitory effects, while FAD had neither effect on ¹⁴CO₂ nor ¹⁴CO₂ plus ¹⁴C-formate production. The results in the present study demonstrate that an α-oxidation enzyme in cucumber is capable of oxidizing palmitic acid via 2-hydroxy- and 2-oxopalmitic acid to produce pentadecanal and CO₂. In contrast to the subcellular 150,000×g _max fraction, the purified α-oxidation enzyme could neither produce formate nor convert ¹⁴C-formate into ¹⁴CO₂, indicating two possible α-oxidation routes in cucumber.