Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.2.1.13 (acetyl-CoA synthetase)
 451 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fatty acids are utilized as a cellular energy source. In the present study, we investigated whether fatty acids could affect axoplasmic transport. Cultured mouse superior cervical ganglion neurons were placed in the glucose-containing medium (145 mM NaCl, 5 mM KCl, 1 mM CaCl(2), 1 mM MgCl(2), 5 mM D-glucose, 10 mM Hepes, pH 7.3, 37 degrees C), and axoplasmic transport of particles in neurites was observed under video-enhanced contrast microscopy. A variety of fatty acids (acetate (C2), caproate (C6), caprylate (C8), caprate (C10), 2-decenoate (C10:1), arachidonate (C20:4); 0.1-1 mM) caused a transient increase in the amount of particles transported in both anterograde and retrograde directions. The increasing effects of fatty acids were dose-dependent. A half-maximum effective dose (ED(50)) for acetate was 0.8 mM, which is similar to the reported K(m) value of acetyl-CoA synthetase for acetate. The ED(50) for caprylate was 28 microM, which is near the K(m) value of acyl-CoA synthetase for medium- and long-chain fatty acids. Application of 5 mM malonate, an inhibitor of the citrate cycle, induced a steady-state decrease in axoplasmic transport, indicating that energy derived from the citrate cycle is required for the maintenance of axoplasmic transport. The increasing effect of acetate (1 mM) on axoplasmic transport was completely abolished by pretreatment with malonate (5 mM), suggesting that acetate produces ATP for axoplasmic transport via the citrate cycle. Alternatively, the effect of caprate (1 mM) was retained after treatment with malonate. Thus, fatty acids except acetate produce ATP probably through both the beta-oxidation pathway and the citrate cycle, increasing axoplasmic transport. Since the effect of fatty acids was transient, certain negative feedback mechanisms might be involved. The removal of glucose from the medium resulted in a low steady-state level of axoplasmic transport. Under such condition, the acetate (1 mM)-induced transient increase in axoplasmic transport remained. Since intracellular ATP must be low under glucose-free condition, intracellular ATP concentrations are unlikely to be involved in the feedback system. Instead, acetyl-CoA or its downstream products in the citrate cycle might lead to feedback inhibition. Application of citrate (5 mM) caused a strong decrease following a transient increase in axoplasmic transport, whereas no other acetyl-CoA product decreased axoplasmic transport. Thus, excessive citrate may be one of factors leading to feedback inhibition of metabolic pathways to arrest and reverse the increase in axoplasmic transport induced by fatty acids.
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PMID:Fatty acids as an energy source for the operation of axoplasmic transport. 1271 Oct 76

The aim of this work was to understand the steps controlling the process of biotransformation of trimethylamonium compounds into L(-)-carnitine by Escherichia coli and the link between the central carbon or primary and the secondary metabolism expressed. Thus, the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA (pyruvate dehydrogenase, acetyl-CoA synthetase, and ATP:acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (isocitrate dehydrogenase) and glyoxylate (isocitrate lyase) cycles, were followed in batch with both growing and resting cells and during continuous cell growth in stirred-tank and high-cell-density membrane reactors. In addition, the levels of carnitine, crotonobetaine, gamma-butyrobetaine, ATP, NADH/NAD(+), and acetyl-CoA/CoA ratios were measured to determine how metabolic fluxes were distributed in the catabolic system. The results provide the first experimental evidence demonstrating the important role of the glyoxylate shunt during biotransformation of resting cells and the need for high levels of ATP to maintain metabolite transport and biotransformation (2.1 to 16.0 mmol L cellular/mmol ATP L reactor h). Moreover, the results obtained for the pool of acetyl-CoA/CoA indicate that it also correlated with the biotransformation process. The main metabolic pathway operating during cell growth in the high cell-density membrane reactor was that related to isocitrate dehydrogenase (during start-up) and isocitrate lyase (during steady-state operation), together with phosphotransacetylase and acetyl-CoA synthetase. More importantly, the link between central carbon and L(-)-carnitine metabolism at the level of the ATP pool was also confirmed.
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PMID:Link between primary and secondary metabolism in the biotransformation of trimethylammonium compounds by escherichia coli. 1459 81

ATP-citrate lyase (Acly) is one of two cytosolic enzymes that synthesize acetyl-coenzyme A (CoA). Because acetyl-CoA is an essential building block for cholesterol and triglycerides, Acly has been considered a therapeutic target for hyperlipidemias and obesity. To define the phenotype of Acly-deficient mice, we created Acly knockout mice in which a beta-galactosidase marker is expressed from Acly regulatory sequences. We also sought to define the cell type-specific expression patterns of Acly to further elucidate the in vivo roles of the enzyme. Homozygous Acly knockout mice died early in development. Heterozygous mice were healthy, fertile, and normolipidemic on both chow and high fat diets, despite expressing half-normal amounts of Acly mRNA and protein. Fibroblasts and hepatocytes from heterozygous Acly mice contained half-normal amounts of Acly mRNA and protein, but this did not perturb triglyceride and cholesterol synthesis or the expression of lipid biosynthetic genes regulated by sterol regulatory element-binding proteins. The expression of acetyl-CoA synthetase 1, another cytosolic enzyme for producing acetyl-CoA, was not up-regulated. As judged by beta-galactosidase staining, Acly was expressed ubiquitously but was expressed particularly highly in tissues with high levels of lipogenesis, such as in the livers of mice fed a high-carbohydrate diet. beta-Galactosidase staining was intense in the developing brain, in keeping with the high levels of de novo lipogenesis of the tissue. In the adult brain, beta-galactosidase staining was in general much lower, consistent with reduced levels of lipogenesis; however, beta-galactosidase expression remained very high in cholinergic neurons, likely reflecting the importance of Acly in generating acetyl-CoA for acetylcholine synthesis. The Acly knockout allele is useful for identifying cell types with a high demand for acetyl-CoA synthesis.
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PMID:ATP-citrate lyase deficiency in the mouse. 1466 65

Data from laboratory-scale sequencing batch reactors operated in an anaerobic-aerobic cycle showed that a low influent phosphorus/chemical oxygen demand (COD) ratio feed favored a glycogen-accumulating metabolism (GAM)-dominated culture and that a high influent phosphorus/COD ratio feed favored a polyphosphate-accumulating metabolism (PAM)-dominated culture. The PAM-dominated culture anaerobically took up acetate approximately 7 times faster than the GAM-dominated culture. Adenosine triphosphate (ATP) balances were performed assuming eight different metabolic scenarios that included the Entner-Doudoroff or the Embden-Myerhof glycolytic pathway, acetyl-coenzyme A (CoA) synthase or the acetate kinase-phospho-transacetylase (AK-PTA) system for acetyl-CoA synthesis, and ATP synthesis or no ATP synthesis during fumarate reduction. The ATP available for transport of acetate into the cell (2) was calculated using these balances. The assumed quantity of ATP produced during fumarate reduction had a relatively small effect on alpha, particularly when PAM was dominant. When GAM was dominant, little or no ATP was available for acetate transport depending on the assumed scenario, and the Embden-Myerhof pathway was more feasible. The value of alpha increased with increasing PAM dominance for all eight metabolic pathways. The maximum calculated alpha value of 0.5 mol ATP/C-mol acetate uptake occurred at maximum PAM dominance and when the Embden-Myerhof pathway was active, when ATP was produced during fumarate reduction, and when the AK-PTA system was active. This value of alpha was higher than previously calculated values with the same metabolic assumptions. An acetate uptake mechanism was suggested that included acetyl-CoA synthetase and direct regeneration of the proton motive force by a proton-translocating pyrophosphatase. Polyphosphate-accumulating metabolism may have a competitive advantage over GAM through a higher anaerobic acetate uptake rate made possible by a greater use of energy for acetate uptake, by use of a different acetate uptake mechanism, or both.
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PMID:Enhanced biological phosphorus removal from wastewater by biomass with different phosphorus contents, Part II: Anaerobic adenosine triphosphate utilization and acetate uptake rates. 1470 9

Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.
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PMID:Fungal ammonia fermentation, a novel metabolic mechanism that couples the dissimilatory and assimilatory pathways of both nitrate and ethanol. Role of acetyl CoA synthetase in anaerobic ATP synthesis. 1472 82

ADP-forming acetyl-CoA synthetase (ACD), the novel enzyme of acetate formation and energy conservation in archaea Acety - CoA + ADP + Pi<==>acetate + ATP CoA), has been studied only in few hyperthermophilic euryarchaea. Here, we report the characterization of two ACDs with unique molecular and catalytic features, from the mesophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. ACD from H. marismortui was purified and characterized as a salt-dependent, mesophilic ACD of homodimeric structure (166 kDa). The encoding gene was identified in the partially sequenced genome of H. marismortui and functionally expressed in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization and refolding in the presence of salts. The ACD catalyzed the reversible ADP- and Pi-dependent conversion of acetyl-CoA to acetate. In addition to acetate, propionate, butyrate, and branched-chain acids (isobutyrate, isovalerate) were accepted as substrates, rather than the aromatic acids, phenylacetate and indol-3-acetate. In the genome of P. aerophilum, the ORFs PAE3250 and PAE 3249, which code for alpha and beta subunits of an ACD, overlap each other by 1 bp, indicating a novel gene organization among identified ACDs. The two ORFs were separately expressed in E. coli and the recombinant subunits alpha (50 kDa) and beta (28 kDa) were in-vitro reconstituted to an active heterooligomeric protein of high thermostability. The first crenarchaeal ACD showed the broadest substrate spectrum of all known ACDs, catalyzing the conversion of acetyl-CoA, isobutyryl-CoA, and phenylacetyl-CoA at high rates. In contrast, the conversion of phenylacetyl-CoA in euryarchaeota is catalyzed by specific ACD isoenzymes.
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PMID:Unusual ADP-forming acetyl-coenzyme A synthetases from the mesophilic halophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. 1534 Jul 86

AMP-forming acetyl-CoA synthetases (ACSs) are ubiquitous in all three domains of life. Here, we report the first characterization of an ACS from a hyperthermophilic organism, from the archaeon Pyrobaculum aerophilum. The recombinant ACS, the gene product of ORF PAE2867, showed extremely high thermostability and thermoactivity at temperatures around 100 degrees C. In contrast to known monomeric or homodimeric mesophilic ACSs, the P. aerophilum ACS was a 610 kDa homooctameric protein, with a significant lower content of thermolabile (Cys, Asn, and Gln) and higher content of charged (Glu, Lys, and Arg) amino acids. Kinetic analyses revealed an unusual broad substrate spectrum for organic acids and an extremely high affinity for acetate (K(m) 3 microM).
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PMID:A novel octameric AMP-forming acetyl-CoA synthetase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. 1564 62

In a series of previous reports it was established by implementing metabolic flux, NMR/MS, and Northern blot analysis that the glyoxylate shunt, the TCA cycle, and acetate uptake by acetyl-CoA synthetase are more active in Escherichia coli BL21 than in Escherichia coli JM109. These differences were accepted as the reason for the differences in the glucose metabolism and acetate excretion of these two strains. Examination of the bacterial metabolism by microarrays and time course Northern blot showed that in addition to the glyoxylate shunt, the TCA cycle and the acetate uptake, other metabolic pathways are active differently in the two strains. These are gluconeogenesis, sfcA shunt, ppc shunt, glycogen biosynthesis, and fatty acid degradation. It was found that in E. coli JM109, acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate rather than by phosphotransacetylase-acetate kinase (Pta-AckA) system which uses acetyl-CoA. The inactivation of the gluconeogenesis enzyme phosphoenolpyruvate synthetase (ppsA), the activation of the anaplerotic sfcA shunt, and low and stable pyruvate dehydrogenase (aceE, aceF) cause pyruvate accumulation which is converted to acetate by pyruvate oxidase B. The behavior of the ppsA, acs, and aceBAK in JM109 was dependent on the glucose supply strategy. When the glucose concentration was high, no transcription of these genes was observed and acetate concentration increased, but at low glucose concentrations these genes were expressed and the acetate concentration decreased. It is possible that there is a major regulatory molecule that controls not only ppsA and aceBAK but also acs. The gluconeogenesis pathway (fbp, pckA, and ppsA) which leads to glycogen accumulation is constitutively active in E. coli BL21 regardless of glucose feeding strategy.
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PMID:Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses. 1580 47

To further enhance the pyruvate productivity by multi-vitamin auxotrophic yeast Torulopsis glabrata, a breeding strategy aiming at decreasing the activity of pyruvate decarboxylase but increasing the activity of acetyl-CoA synthetase was developed based on analysis of pyruvate-related metabolic pathways. Nitrosoguanidine mutagenized cells of T. glabrata WSH-IP303 were screened for mutants that require acetate for complete growth on glucose minimum medium. A mutant, T. glabrata CCTCC M202019, produced pyruvate 21% higher than that of the parent strain and was genetically stable in flask cultures, was selected as a working strain. To elucidate the metabolic changes that led to the increase of pyruvate production, the activities of enzymes that involved in pyruvate-related metabolic pathways of the mutant and the parent strain were determined. Enzymatic analysis revealed that, compared with the parent strain WSH-IP303, the activity of pyruvate decarboxylase of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase of the latter increased by 103.5% or 57.4%, respectively, in the presence or absence of acetate. When 6 g/L sodium acetate was added to the medium, pyruvate production by the mutant strain CCTCC M202019 reached 68.7 g/L at 62 h (yield on glucose, 0.651 g/g) in fermentations performed in a 7-L jar fermentor, indicating the shortage of cytosolic acetyl-CoA resulted from the disruption of pyruvate decarboxylase was properly compensated by the increase of the activity of acetyl-CoA synthetase.
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PMID:[Application of a metabolic-pathway-analysis based breeding strategy enhances the production of pyruvate by Torulopsis glabrata]. 1584 68

We established a novel enzyme-catalyzed poly(3-hydroxybutyrate) [P(3HB)] synthesis system capable of recycling CoA on the basis of the P(3HB) biosynthetic pathway in Ralstonia eutropha. The system includes purified beta-ketothiolase (PhaA), NADPH-dependent acetoacetyl-CoA reductase (PhaB), PHA synthase (PhaC), acetyl-CoA synthetase (Acs) and glucose dehydrogenase (GDH). In this system, acetyl-CoA was synthesized from acetate and CoA by Acs and ATP, and then two molecules of acetyl-CoA were condensed by PhaA to synthesize acetoacetyl-CoA, which was converted to (R)-3-hydroxybutyryl-CoA (3HBCoA) by PhaB and NADPH. The 3HBCoA was polymerized by PhaC and converted to P(3HB). In this system, the CoA molecules that were released during the condensation and polymerization reactions catalyzed by PhaA and PhaC, respectively, were reused successfully for the synthesis of acetyl-CoA. In addition, NADPH, which was consumed in the reduction of acetoacetyl-CoA, was regenerated by the action of GDH. In this system, the yield of P(3HB) synthesized from acetate as the substrate was 5.6 mg in a 5-ml reaction mixture, and the weight-average molecular weight and polydispersity were 6.64 x 10(6) and 1.36, respectively. Furthermore, CoA was reused at least 26 times, and NADPH was also regenerated at least 26 times during 24 h of reaction.
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PMID:Enzyme-catalyzed poly(3-hydroxybutyrate) synthesis from acetate with CoA recycling and NADPH regeneration in Vitro. 1623 16


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