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)

Saccharomyces cerevisiae T23C (pda1::Tn5ble) is an isogenic gene replacement mutant of the wild-type strain S. cerevisiae T23D. The mutation causes a complete loss of pyruvate dehydrogenase activity. Pyruvate metabolism in this pyruvate-dehydrogenase-negative (Pdh-) strain was investigated in aerobic glucose-limited chemostat cultures, grown at a dilution rate of 0.10 h-1, and compared with the metabolism in the isogenic wild-type strain. Under these conditions, growth of the Pdh- strain was fully respiratory. Enzyme activities in cell-free extracts indicated that the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase could provide a functional bypass of the pyruvate dehydrogenase complex. Since this metabolic sequence involves ATP hydrolysis in the acetyl-CoA synthetase reaction, a negative effect of the pda1::Tn5ble mutation on the growth efficiency was anticipated. Indeed, the biomass yield of the Pdh- strain [0.44 g biomass (g glucose)-1] was significantly lower than that of wild-type S. cerevisiae [0.52 g biomass (g glucose)-1]. The effect of the mutation on biomass yield could be quantitatively explained in terms of a lower ATP yield from glucose catabolism and an increased ATP requirement for the synthesis of acetyl-CoA used in anabolism. Control experiments showed that the pda1::Tn5ble mutation did not affect biomass yield in ethanol-limited chemostat cultures. The results support the view that, during aerobic glucose-limited growth of S. cerevisiae at low growth rates, the pyruvate dehydrogenase complex accounts for the major part of the pyruvate flux. Moreover, it is concluded that hydrolysis of pyrophosphate formed in the acetyl-CoA synthetase reaction does not contribute significantly to energy transduction in this yeast. Respiratory-deficient cells did not contribute to glucose metabolism in the chemostat cultures and were probably formed upon plating.
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PMID:Energetic aspects of glucose metabolism in a pyruvate-dehydrogenase-negative mutant of Saccharomyces cerevisiae. 801 82

The mutant gene coding for a proline-activating domain (grs2-pro) was cloned and sequenced from Bacillus brevis Nagano, BII-3 strain, which produces gramicidin S synthetase 2 defective in proline-activation. By comparison of the nucleotide sequence with the wild-type sequence, a single point mutation was found at the 2609th guanine, which was replaced with adenine, resulting in the change of the 870th glycine to glutamic acid. Homology search for the deduced amino acid sequence of grs2-pro gene revealed that the 870th glycine was conserved in adenylate-forming enzymes, and its flanking sequence was highly conserved among the aminoacyl adenylate-forming enzymes, such as antibiotic peptide synthetases: gramicidin S synthetase 1 and 2 (GS1, GS2), tyrocidine synthetase 1 (TS1), and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS); and other aminoacyl adenylation enzymes: alpha-aminoadipate reductase (LYS2), EntF, and AngR. On the other hand, this flanking sequence was not conserved in the other adenylate-forming enzymes lacking amino acid activation, such as acetyl-CoA synthetase, long-chain acyl-CoA synthetase, luciferase, and 4-coumarate CoA ligase. Single base substitutions at the 870th GGG codon were carried out by oligonucleotide site-directed mutagenesis. Four mutagenized clones were isolated, containing grs2-pro genes which exchange 870-Gly for alanine, valine, arginine, and tryptophan. The translated products from these clones could scarcely catalyze proline-dependent ATP-32PPi exchange reaction. The coil structure of 870-Gly region was lost in the mutants. These results suggest that the 870-Gly residue of grs2-pro protein is essential for aminoacyl-adenylation in the antibiotic peptide synthetase family.
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PMID:Effect of single base substitutions at glycine-870 codon of gramicidin S synthetase 2 gene on proline activation. 827 62

Prodigiosin 25-C had little effect on DNA, RNA, and protein synthesis, and cellular ATP content, but the drug markedly inhibited the incorporation of acetate into lipid fractions. Under the same conditions, the incorporation of other lipid precursors including glycerol, mevalonate, palmitate, and oleate was not affected. A decrease in the incorporation of acetate was not due to the inhibition of fatty acid biosynthesis, because prodigiosin 25-C did not affect the activity of acetyl-CoA synthetase, acetyl-CoA carboxylase or fatty acid synthase in cell-free assay systems prepared from rat liver cytosol. In contrast, prodigiosin 25-C strongly inhibited the rapid uptake of acetate into acid-soluble fraction in intact cells. The results suggest that prodigiosin 25-C specifically perturbs the permeation of acetate through plasma membranes.
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PMID:Prodigiosin 25-C perturbs permeation of acetate in a cultured cell line. 853 81

Giardia lamblia, an amitochondriate eukaryote, contains acetyl-CoA synthetase (ADP-forming), an enzyme known only from one other eukaryote (Entamoeba histolytica) and a few anaerobic prokaryotes. The enzyme has been purified about 350-fold. The activity in the direction of acetate formation was dependent on ADP and inorganic phosphate. The reverse reaction could not be detected. Succinyl-CoA, propionyl-CoA and dADP were utilized with lower efficiency. The enzyme did not utilize AMP plus PPi thus differs from the broadly distributed acetyl-CoA synthetase (AMP-forming). The enzyme is responsible for acetate production accompanied by ATP generation, thus plays an important role in G. lamblia metabolism.
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PMID:Purification and characterization of the acetate forming enzyme, acetyl-CoA synthetase (ADP-forming) from the amitochondriate protist, Giardia lamblia. 855 9

High rates of methanogenesis from acetate and ATP were observed from cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix (Methanosaeta) thermophila strain CALS-1 when cultures were grown in a pH auxostat fed with acetic acid. Specific methanogenic activities ranged from 50-300 nmol min-1 (mg protein)-1, which was comparable to those for whole cells. In contrast to results with Methanosarcina spp., the reaction did not require high levels of H2 in the headspace. CO was inhibitory to methanogenesis from acetate. The inhibition by CO and the lack of effect of H2 on methanogenesis from acetate resemble previous results with whole cells of CALS-1. Protein concentrations in extracts > 5 mg/ml were required for good activity, and the optimum temperature for the methanogenesis was near 65° C. ATP was required in substrate quantities and was converted mainly to AMP. The maximum CH4/ATP stoichiometry obtained was near 1.0, consistent with acetate activation using an acetyl-CoA synthetase mechanism that converts ATP to AMP and pyrophosphate. Methanogenesis in extracts was inhibited by bromoethane sulfonate and cyanide, indicating the involvement of methylcoenzyme M methylreductase and a carbon monoxide dehydrogenase complex with methanogenesis from acetate. These results are consistent with acetyl-coenzyme A (CoA) as the form of activated acetate involved in methanogenesis from acetate in strain CALS-1, but no activity could be obtained from extracts using acetyl-CoA as a substrate.
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PMID:Methanogenesis from acetate by cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix thermophila CALS-1 882 51

Acetyl-CoA synthetase (ADP-forming) is an enzyme in Archaea that catalyzes the formation of acetate from acetyl-CoA and couples this reaction with the synthesis of ATP from ADP and Pi (acetyl-CoA + ADP + Pi --> acetate + ATP + CoA) [Schifer, T., Selig, M. & Schonheit, P. (1993) Arch. Microbiol. 159, 72-83]. The enzyme from the anaerobic hyperthermophile Pyrococcus furiosus was purified 96-fold with a yield of 20% to apparent electrophoretic homogeneity. The oxygen-stable enzyme had an apparent molecular mass of 145 kDa and was composed of two subunits with apparent molecular masses of 47 kDa and 25 kDa, indicating an alpha2beta2 structure. The N-terminal amino acid sequences of both subunits were determined; they do not show significant identity to other proteins in databases. The purified enzyme catalyzed the reversible conversion of acetyl-CoA, ADP and Pi to acetate, ATP and CoA. The apparent Vmax value in the direction of acetate formation was 18 U/mg (55 degrees C), the apparent Km values for acetyl-CoA, ADP and Pi were 17 microM, 60 microM and 200 microM, respectively. ADP and Pi could not be replaced by AMP and PPi, defining the enzyme as an ADP-forming rather than an AMP-forming acetyl-CoA synthetase. The apparent Vmax value in the direction of acetyl-CoA formation was about 40 U/mg (55 degrees C), and the apparent Km values for acetate, ATP and CoA were 660 microM, 80 microM and 30 microM, respectively. The purified enzyme was not specific for acetyl-CoA or acetate, in addition to acetyl-CoA (100%), the enzyme accepts propionyl-CoA (110%) and butyryl-CoA (92%), and in addition to acetate (100%), the enzyme accepts propionate (100%), butyrate (92%), isobutyrate (79%), valerate (36%) and isovalerate (34%), indicating that the enzyme functions as an acyl-CoA synthetase (ADP-forming) with a broad substrate spectrum. Succinate, phenylacetate and indoleacetate did not serve as substrates for the enzyme (<3%). In addition to ADP (100%), GDP (220%) and IDP (250%) were used, and in addition to ATP (100%), GTP (210%) and ITP (320%) were used. Pyrimidine nucleotides were not accepted. The enzyme was dependent on Mg2+, which could be partly substituted by Mn2+ and Co2+. The pH optimum was pH 7. The enzyme has a temperature optimum at 90 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions. The enzyme was stabilized against heat inactivation by salts. In the presence of KCI (1 M), which was most effective, the enzyme did not loose activity after 2 h incubation at 100 degrees C.
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PMID:Purification and properties of acetyl-CoA synthetase (ADP-forming), an archaeal enzyme of acetate formation and ATP synthesis, from the hyperthermophile Pyrococcus furiosus. 911 24

Purified recombinant poly(hydroxyalkanoic acid), PHA, synthase from Chromatium vinosum was used to examine in vitro poly(3-hydroxybutyric acid) (P(3HB)) formation. In combination with purified propionyl-coenzyme A transferase of Clostridium propionicum a two-enzyme in vitro P(3HB) biosynthesis system was established which allowed the synthesis of P(3HB) from free D-(-)-3-hydroxybutyric acid as substrate. The coenzyme A residue for the activation of this hydroxyacid was provided by acetyl-coenzyme A. By adding acetyl-coenzyme A synthetase to this system, a three-enzyme in vitro P(3HB) biosynthesis system was established. Coenzyme A that was released during the polymerization reaction was coupled to acetate which again served as the coenzyme A donor for the activation of 3-hydroxybutyric acid. The energy for the in vitro P(3HB) synthesis was provided by ATP hydrolyses resulting in acetyl-coenzyme A synthesis catalyzed by the acetyl coenzyme A synthetase. In this way the in vitro synthesis of P(3HB) became independent of the consumption of the expensive coenzyme A. By this procedure a handy system is available to produce in vitro PHA on a semipreparative scale.
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PMID:In vitro synthesis of poly(3-hydroxybutyric acid) by using an enzymatic coenzyme A recycling system. 983 44

The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.
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PMID:Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation. 988 36

Biochemical and genetic evidence is presented to demonstrate that the prpE gene of Salmonella typhimurium encodes propionyl-CoA synthetase, an enzyme required for the catabolism of propionate in this bacterium. While prpE mutants used propionate as carbon and energy source, prpE mutants that lacked acetyl-CoA synthetase (encoded by acs) did not, indicating that Acs can compensate for the lack of PrpE in prpE mutants. Cell-free extracts enriched for PrpE catalysed the formation of propionyl-CoA in a propionate-, ATP-, Mg2+- and HS-CoA dependent manner. Acetate substituted for propionate in the reaction at 48% the rate of propionate; butyrate was not a substrate for PrpE. The propionyl-CoA synthetase activity of PrpE was specific for ATP. GTP, ITP, CTP and TTP were not used as substrates by the enzyme. UV-visible spectrophotometry, HPLC and MS data demonstrated that propionyl-CoA was the product of the reaction catalysed by PrpE.
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PMID:The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase. 1041 Dec 65

Acetyl-coenzyme A (acetyl-CoA) synthetase (ADP forming) represents a novel enzyme in archaea of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) --> acetate + ATP + CoA). Two isoforms of the enzyme have been purified from the hyperthermophile Pyrococcus furiosus. Isoform I is a heterotetramer (alpha(2)beta(2)) with an apparent molecular mass of 145 kDa, composed of two subunits, alpha and beta, with apparent molecular masses of 47 and 25 kDa, respectively. By using N-terminal amino acid sequences of both subunits, the encoding genes, designated acdAI and acdBI, were identified in the genome of P. furiosus. The genes were separately overexpressed in Escherichia coli, and the recombinant subunits were reconstituted in vitro to the active heterotetrameric enzyme. The purified recombinant enzyme showed molecular and catalytical properties very similar to those shown by acetyl-CoA synthetase (ADP forming) purified from P. furiosus.
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PMID:Acetyl coenzyme A synthetase (ADP forming) from the hyperthermophilic Archaeon pyrococcus furiosus: identification, cloning, separate expression of the encoding genes, acdAI and acdBI, in Escherichia coli, and in vitro reconstitution of the active heterotetrameric enzyme from its recombinant subunits. 1048 38


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