Studies on the hydrolysis and utilization of long chain acyl CoA thioesters by liver microsomes

Acyl CoA thioester “hydrolase” activity exhibited by liver microsomal preparations using a spectrophotometric assay for released CoA has been shown to be a composite of several enzymatic reactions. Pig liver microsomes form a considerable amount of triacylglycerol from endogenous precursors, with essentially all acyl groups being incorporated into the 1,3 positions. Rat and rabbit liver microsomes form very little triacylglycerol under the same conditions. The true acyl CoA hydrolase activity of pig liver microsomes is relatively insensitive to diisopropylfluorophosphate (DFP), whereas this compound strongly inhibits the rat and rabbit enzymes. The hydrolase remaining in the liver microsomes after DFP treatment is rapidly inactivated when incubated in the presence of substrate.     

The Pathophysiological Role of CoA

The importance of coenzyme A (CoA) as a carrier of acyl residues in cell metabolism is well understood. Coenzyme A participates in more than 100 different catabolic and anabolic reactions, including those involved in the metabolism of lipids, carbohydrates, proteins, ethanol, bile acids, and xenobiotics. However, much less is known about the importance of the concentration of this cofactor in various cell compartments and the role of altered CoA concentration in various pathologies. Despite continuous research on these issues, the molecular mechanisms in the regulation of the intracellular level of CoA under pathological conditions are still not well understood. This review summarizes the current knowledge of (a) CoA subcellular concentrations; (b) the roles of CoA synthesis and degradation processes; and (c) protein modification by reversible CoA binding to proteins (CoAlation). Particular attention is paid to (a) the roles of changes in the level of CoA under pathological conditions, such as in neurodegenerative diseases, cancer, myopathies, and infectious diseases; and (b) the beneficial effect of CoA and pantethine (which like CoA is finally converted to Pan and cysteamine), used at pharmacological doses for the treatment of hyperlipidemia.     

The Structure of LiuC,a 3‐Hydroxy‐3‐Methylglutaconyl CoA Dehydratase Involved in Isovaleryl‐CoA Biosynthesis in Myxococcus xanthus,Reveals Insights into Specificity and Catalysis

Myxobacteria are able to produce the important metabolite isovaleryl coenzyme A by a route other than leucine degradation. The first step into this pathway is mediated by LiuC, a member of the 3‐methylglutaconyl CoA hydratases (MGCH). Here we present crystal structures refined to 2.05 and 1.1 Å of LiuC in the apo form and bound to coenzyme A, respectively. By using simulated annealing we modeled the enzyme substrate complex and identified residues potentially involved in substrate binding, specificity, and catalysis. The dehydration of 3‐hydroxy‐3‐methylglutaconyl CoA to 3‐methylglutaconyl CoA catalyzed by LiuC involves Glu112 and Glu132 and likely employs the typical crotonase acid‐base mechanism. In this, Tyr231 and Arg69 are key players in positioning the substrate to enable catalysis. Surprisingly, LiuC shows higher sequence and structural similarity to human MGCH than to bacterial forms, although they convert the same substrate. This study provides structural insights into the alternative isovaleryl coenzyme A biosynthesis pathway and might open a path for biofuel research, as isovaleryl‐CoA is a source for isobutene, a precursor for renewable fuels and chemicals.     

芳香羧酸脱羧-偶联反应研究

芳香族羧酸脱羧-偶联反应能够有效构建C—C或C-杂原子键,在天然活性物质、药物中间体以及特殊材料合成等方面具有重要意义,并且该方法具有很好的区域选择性、原子经济性及环境友好性等特点.近年来以芳香羧酸为基本原料的脱羧-偶联反应得到深入研究,新颖催化剂不断涌现,反应条件不断完善,为该类反应的应用提供了理论依据.从偶联反应的...     

杂多酸催化剂在芳酮合成中的应用

综述了杂多酸及其盐在合成芳酮的Friedel-Crafts(F-C)酰化反应和Fries重排中的应用,并与其它类型的催化剂作了比较。结果表明,杂多酸(盐)是一类性能优良的合成芳酮的催化剂,其催化性能优于沸石、分子筛等固体酸催化剂。     

Generation of Cannabigerolic Acid Derivatives and Their Precursors by Using the Promiscuity of the Aromatic Prenyltransferase NphB

NphB is an aromatic prenyltransferase with high promiscuity for phenolics including flavonoids, isoflavonoids, and plant polyketides. It has been demonstrated that cannabigerolic acid is successfully formed by the reaction catalysed by NphB using geranyl diphosphate and olivetolic acid as substrates. In this study, the substrate specificity of NphB was further determined by using olivetolic acid derivatives as potential substrates for the formation of new synthetic cannabinoids. The derivatives differ in the hydrocarbon chain attached to C6 of the core structure. We performed in silico experiments, including docking of olivetolic acid derivatives, to identify differences in their binding modes. Substrate acceptance was predicted. Based on these results, a library of olivetolic acid derivatives was constructed and synthesized by using different organic synthetic routes. Conversion was monitored in in vitro assays with purified NphB versions. For the substrates leading to a high conversion olivetolic acid-C8, olivetolic acid-C2 and 2-benzyl-4,6-dihydroxybenzoic acid, the products were further elucidated and identified as cannbigerolic acid derivatives. Therefore, these substrates show potential to be adapted in cannabinoid biosynthesis.     

单酯基多环芳香酸的合成及热分析

以4-正丙基,4-正戊基取代的双环己基甲酸、联苯甲酸和环己基苯甲酸以及对羟基苯甲醛为原料,用一次酯化、一次氧化的方法(酯化氧化法),合成了4个单酯基芳香酸类化合物:4-〔4-(反-4-丙基环己基)苯酰氧基〕苯甲酸,4〔-反-4-(反-4-丙基环己基)环己酰氧基〕苯甲酸,4-〔反-4-(反-4-戊基环己基)环己酰氧基〕苯甲酸和4-〔4-(4-戊基苯基)苯酰氧基〕苯甲酸,总产率分别为74%,76%,72%和71%。产物结构用IR,MS,1HNMR及元素分析进行了表征,DSC及偏光显微镜测定表明,4个目标化合物具有热致液晶性。     

4-Arylthiosemicarbazide Derivatives as Toxoplasmic Aromatic Amino Acid Hydroxylase Inhibitors and Anti-inflammatory Agents

Approximately one-third of the human population is infected with the intracellular cosmopolitan protozoan Toxoplasma gondii (Tg), and a specific treatment for this parasite is still needed. Additionally, the increasing resistance of Tg to drugs has become a challenge for numerous research centers. The high selectivity of a compound toward the protozoan, along with low cytotoxicity toward the host cells, form the basis for further research, which aims at determining the molecular targets of the active compounds. Thiosemicarbazide derivatives are biologically active organic compounds. Previous studies on the initial preselection of 58 new 4-arylthiosemicarbazide derivatives in terms of their anti-Tg activity and selectivity made it possible to select two promising derivatives for further research. One of the important amino acids involved in the proliferation of Tg and the formation of parasitophorous vacuoles is tyrosine, which is converted by two unique aromatic amino acid hydroxylases to levodopa. Enzymatic studies with two derivatives (R: para-nitro and meta-iodo) and recombinant aromatic amino acid hydroxylase (AAHs) obtained in the E. coli expression system were performed, and the results indicated that toxoplasmic AAHs are a molecular target for 4-arylthiosemicarbazide derivatives. Moreover, the drug affinity responsive target stability assay also confirmed that the selected compounds bind to AAHs. Additionally, the anti-inflammatory activity of these derivatives was tested using THP1-Blue™ NF-κB reporter cells due to the similarity of the thiosemicarbazide scaffold to thiosemicarbazone, both of which are known NF-κB pathway inhibitors.     

Lipid products formed during desaturation of [1-14C] stearyl CoA by hen liver microsomes

A number of lipid products are formed during the desaturation of stearyl CoA by hen liver microsomes. This article presents an analysis of the products formed when [1-¹⁴C] stearyl CoA is incubated with hen liver microsomes for various time periods. [1-¹⁴C] Oleyl CoA was the first radioactive unsaturated product formed. Synthesis of phospholipids containing [1-¹⁴C] oleic acid occurs only after the desaturase activity has begun to decline. The specific radioactivity of [1-¹⁴C] oleyl CoA was similar to the specific radioactivity of [1-¹⁴C] stearyl CoA at all time periods tested. The specific radioactivities of [1-¹⁴C] oleic acid and phospholipids containing [1-¹⁴C] oleic acid were much lower than that of the [1-¹⁴C] stearyl CoA.     

Inducing Effect of Clofibric Acid on Stearoyl‐CoA Desaturase in Intestinal Mucosa of Rats

Fibrates have been reported to elevate the hepatic proportion of oleic acid (18:1n‐9) through inducing stearoyl‐CoA desaturase (SCD). Despite abundant studies on the regulation of SCD in the liver, little is known about this issue in the small intestine. The present study aimed to investigate the effect of clofibric acid on the fatty acid profile, particularly monounsaturated fatty acids (MUFA), and the SCD expression in intestinal mucosa. Treatment of rats with a diet containing 0.5 % (w/w) clofibric acid for 7 days changed the MUFA profile of total lipids in intestinal mucosa; the proportion of 18:1n‐9 was significantly increased, whereas those of palmitoleic (16:1n‐7) and cis‐vaccenic (18:1n‐7) acids were not changed. Upon the treatment with clofibric acid, SCD was induced and the gene expression of SCD1, SCD2, and fatty acid elongase (Elovl) 6 was up‐regulated, but that of Elovl5 was unaffected. Fat‐free diet feeding for 28 days increased the proportions of 16:1n‐7 and 18:1n‐7, but did not effectively change that of 18:1n‐9, in intestinal mucosa. Fat‐free diet feeding up‐regulated the gene expression of SCD1, but not that of SCD2, Elovl6, or Elovl5. These results indicate that intestinal mucosa significantly changes its MUFA profile in response to challenges by clofibric acid and a fat‐free diet and suggest that up‐regulation of the gene expression of SCD along with Elovl6 is indispensable to elevate the proportion of 18:1n‐9 in intestinal mucosa.     

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

Triacylglycerol (TAG) is the major storage lipid in most terrestrial plants and microalgae, and has great nutritional and industrial value. Since the demand for vegetable oil is consistently increasing, numerous studies have been focused on improving the TAG content and modifying the fatty‐acid compositions of plant seed oils. In addition, there is a strong research interest in establishing plant vegetative tissues and microalgae as platforms for lipid production. In higher plants and microalgae, TAG biosynthesis occurs via acyl‐CoA‐dependent or acyl‐CoA‐independent pathways. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step in the acyl‐CoA‐dependent biosynthesis of TAG, which appears to represent a bottleneck in oil accumulation in some oilseed species. Membrane‐bound and soluble forms of DGAT have been identified with very different amino‐acid sequences and biochemical properties. Alternatively, TAG can be formed through acyl‐CoA‐independent pathways via the catalytic action of membrane‐bound phospholipid:diacylglycerol acyltransferase (PDAT). As the enzymes catalyzing the terminal steps of TAG formation, DGAT and PDAT play crucial roles in determining the flux of carbon into seed TAG and thus have been considered as the key targets for engineering oil production. Here, we summarize the most recent knowledge on DGAT and PDAT in higher plants and microalgae, with the emphasis on their physiological roles, structural features, and regulation. The development of various metabolic engineering strategies to enhance the TAG content and alter the fatty‐acid composition of TAG is also discussed.     

The effect of protein deficiency on some rat liver lipid metabolic enzymes and CoA

Two groups of male Wistar rats were fed normal (i.e., 18%) and protein-free diets, respectively, for 7 weeks. In vivo incorporation of [1-¹⁴C] acetate into palmitic, stearic, oleic, and arachidonic acids by the liver was reduced in the protein-deficient rats. In vitro incubation of liver microsomes with labeled palmitate or linoleate revealed no change in the specific activities of chain elongating or desaturating enzymes. Protein deficiency resulted in a decrease in specific activity of short chain acyl-CoA synthetase and in total CoA, accompanied by the virtual disappearance of acyl-CoA and an increase in free CoA. Furthermore, there was less microsomal fatty acid synthetase and mitochondrial β-hydroxybutyrate dehydrogenase activity. These results are discussed in relation to fatty acid synthesis and the changes in liver fatty acid composition.     

4-氨基-1,2,4-三氮唑并芳香醛类希夫碱的合成与表征

三氮唑环为抗真菌药物的基本药效基团,为探索芳香醛缩氨基三氮唑的合成条件,以4-氨基-1,2,4-三氮唑与芳香醛为原料,在冰醋酸催化下合成了9种芳香醛类希夫碱,收率71.6%~92.3%,并通过元素分析、IR和1H NMR确定了目标化合物的结构。其中几种鲜见文献报道。同时讨论了影响反应收率的因素。     

Aqueous Asymmetric Mukaiyama Aldol Reaction Catalyzed by Chiral Gallium Lewis Acid with Trost‐Type Semi‐Crown Ligands

The combination of Ga(OTf)₃ with chiral semi‐crown ligands ( 1a – e ) generates highly effective chiral gallium Lewis acid catalysts for aqueous asymmetric aldol reactions of aromatic silyl enol ethers with aldehydes. A ligand‐acceleration effect was observed. Water is essential for obtaining high diastereoselectivity and enantioselectivity. The p‐phenyl substituent in aromatic silyl enol ether ( 2 h ) plays an important role and increases the enantioselectivity up to 95% ee. Although aliphatic silyl enol ethers provided low enantioselectivities and silylketene acetal is easily hydrolyzed in aqueous alcohol, the aldol reactions of silylketene thioacetal ( 12 ) with aldehydes in the presence of gallium‐Lewis acid catalysts give the β‐hydroxy thioester with reasonable yields and high diastereo‐ (up to 99 : 1) and enantioselectivities (up to 96% ee).     

Influence of diet on the kinetic behavior of hepatic carnitine palmitoyltransferase I toward different acyl CoA esters

The influence of diet on the kinetics of the overt form of rat liver mitochondrial carnitine palmitoyltransferase (CPT I; EC 2.3.1.21) was studied using rats fed either a low-fat diet (3% w/w fat), or diets which were supplemented with either olive oil (OO), safflower oil (SO) or menhaden (fish) oil (MO) to 20% w/w of fat (high fat diets). When animals were fed each of these four diets for 10 days, the order of the apparent maximal activity (V_max) of CPT I toward various individual fatty acyl CoA, when measured under a fixed molar ratio of acyl CoA/albumin, was 16∶1n−7>18∶1n−9>18∶2n−6>16∶0>22∶6n−3, and was thus not affected by the fat composition of the diet. However, in all but one case, the SO and MO diets elicited a higher V_max for each substrate than either the LF diet or the high fat OO diet. The apparent K _0.5 for the different acyl CoA esters was generally lowest in LF-fed animals, and highest in those fed the high-fat SO diet. Moreover, when compared with the situation of animals fed high-fat diets, the K _0.5 values of CPT I in LF-fed animals for palmitoyl CoA and oleoyl CoA were low. This possession by CPT I of a high “affinity” toward these nonessential fatty acyl CoAs, but a lower “affinity” toward linoleoyl CoA, the ester of an essential fatty acid, may enable this latter fatty acid to be spared from oxidation when its concentration in the diet is low. The data also emphasize that palmitoleoyl CoA, if available in the diet, is likely to be utilized by CPT I at a high rate.     

Use of Methylmalonyl‐CoA Epimerase in Enhancing Crotonase Stereoselectivity

The use of methylmalonyl‐CoA epimerase (MCEE) to improve stereoselectivity in crotonase‐mediated biocatalysis is exemplified by the coupling of MCEE, crotonyl‐CoA carboxylase reductase and carboxymethylproline synthase in a three‐enzyme one‐pot sequential synthesis of functionalised C‐5 carboxyalkylprolines starting from crotonyl‐CoA and carbon dioxide.     

Rat hepatocyte plasma membrane acyl:CoA synthetase activity

The presence of long chain acyl:CoA synthetases in mammalian microsomes and mitochondria has been established previously by Aas (Biochim. Biophys. Acta 231, 32–47 [1971]). The presence of a plasma membrane-associated enzyme was investigated in rat hepatocyte plasma membranes, where an enzyme exhibiting high activity and with a preferred substrate of 18-carbon chain length was discovered. The results are consistent with the presence of a single enzyme. The effect of the degree of unsaturation of the fatty acid substrates was not as pronounced as that arising from the length of the carbon chain. The pattern of substrate preference of the enzyme was ω3 polyenoic fatty acids >ω6 polyenoic acids >ω9 monoenoic acids > saturated acids. This may relate to the similar substrate preference pattern exhibited by the fatty acyl desaturase enzymes. The role played by long chain acyl:CoA synthetase in hepatocyte metabolism is uncertain, but it may relate to the incorporation of polyenoic fatty acids from the circulation into cell membranes and the trapping of other fatty acids within the cell for further metabolism.     

Biosynthesis of Trehangelin in Polymorphospora rubra K07–0510: Identification of Metabolic Pathway to Angelyl‐CoA

Trehangelins are trehalose angelates discovered from endophytic actinomycete Polymorphospora rubra K07‐0510. We identified the trehangelin biosynthetic gene cluster, including genes that encode a glycoside hydrolase‐like protein (thgC), α‐amylase (thgD), 3‐ketoacyl‐ACP synthase III (thgI), 3‐ketoacyl‐ACP reductase (thgK), enoyl‐CoA hydratase (thgH) and acyl transferase (thgJ). Heterologous expression of thgH, thgI, thgJ and thgK confirmed the importance of these genes in the biosynthesis of trehangelin A. Enzymatic activity studies showed that ThgI catalyses the condensation of acetyl‐CoA and methylmalonyl‐CoA to 2‐methylacetoacetyl‐CoA (MAA‐CoA), ThgK catalyses NADPH‐dependent reduction of MAA‐CoA to 3‐hydroxy‐2‐methylbutyryl‐CoA (HMB‐CoA) and ThgH catalyses the dehydration of HMB‐CoA to angelyl‐CoA (AN‐CoA). This is the first report on the elucidation of the enzymatic formation of AN‐CoA.     

Saturated fatty acids >C20 are not activated by acid:CoA ligase in rat brain or liver?

The formation of long-chain saturated acyl-[³H]CoA and [1-¹⁴C] acyl-[³H]CoA by rat brain microsomes and rat liver was examined. Acyl-CoA formation was markedly decreased as fatty-acid chain length increased from C₁₆ to C₂₀. No biosynthesis of behenyl-[³H] CoA or [1-¹⁴C] lignoceryl-[³H] CoA was observed. The results suggest that long-chain saturated fatty acids >20 carbons in length are not activated by acid:CoA ligase to form acyl-CoA.     

Comparative Study on the Syntheses and Properties of Three N-lauroyl Aromatic Amino Acid Surfactants

Three N-lauroyl aromatic amino acid surfactants, namely, sodium N-lauroyl phenylalaninate (SLP), sodium N-lauroyl tyrosinate, and sodium N-lauroyl tryptophanate (SLTr), with aromatic group structures were synthesized via methyl ester method. Mass spectrometry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance were applied for the structural representation of products. A comparative study of the three surfactants in terms of surface, foam, and emulsifying properties and decontamination performance was conducted. Results demonstrated that aromatic structure significantly influenced the properties of the three surfactants. SLTr had the best surface performance and foam property, whereas SLP had the best emulsifying property. All three surfactants exhibited strong decontamination effects on protein-soiled swatches.