Purification and properties of acetate kinase from Clostridium thermoaceticum

Schaupp, Annabella; Ljungdahl, Lars G.

doi:10.1007/bf00446312

Cited by 66 publications

(42 citation statements)

References 25 publications

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“…Alcohol dehydrogenase activity remained unchanged between 58 and 70°C. Acetate kinase activity was not detected at 70°C, indicating inactivation at this high temperature, an observation which has also been made with Clostridium thermocellum, Thermoanaerobacterium brockii, and Clostridium thermoaceticum (22,32). Due to the enzyme's involvement in energy metabolism, inactivation of acetate kinase might be responsible for the decrease in cell growth of DSM 5809 at 70°C.…”

Section: Resultsmentioning

confidence: 69%

Elucidation of Enzymes in Fermentation Pathways Used by Clostridium thermosuccinogenes Growing on Inulin

Sridhar

Eiteman

Wiegel

2000

Appl Environ Microbiol

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Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by Clostridium thermosuccinogenes DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70°C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58°C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70°C. No acetate kinase activity was observed at 70°C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58°C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70°C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70°C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature.Clostridium thermosuccinogenes is a strictly anaerobic sporeforming gram-positive bacterium that can ferment inulin to succinate and acetate as major products and to lactate, ethanol, formate, hydrogen, and carbon dioxide as minor products (7). Inulin is a carbohydrate found in the roots or tubers of some plants, consisting of 20 to 30 fructose molecules connected to a terminal glucose residue by a ␤ (231) linkage. High concentrations of inulin are found in the roots of Jerusalem artichoke (80% [weight/dry weight]), chicory (75 % [wt/ wt]), and dahlia (72% [wt/wt]) (16).Succinic acid is a four-carbon aliphatic dicarboxylic acid having a pK a1 of 4.2 and a pK a2 of 5.6 which has applications in the manufacture of specialty chemicals and in agriculture, food, medicine, textiles, plating, and waste gas scrubbing (41). Industrially, succinic acid is currently produced by hydrogenation of maleic anhydride to succinic anhydride followed by hydration to succinic acid (5, 41). Succinic acid can be produced by many anaerobic microorganisms, usually near neutral pH (5). For example, one method to produce succinic acid microbially employs the strict anaerobe Anaerobiospirillum succiniciproducens (5, 10, 11, 24). Under optimal conditions, these bacteria produce succinic acid from glucose with a yield of 87% and a final concentration of 35 g/liter (11, 24). Recently, Guettler et al. (15) isolated the facultatively anaerobic gram-negativ...

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Section: Resultsmentioning

confidence: 69%

Elucidation of Enzymes in Fermentation Pathways Used by Clostridium thermosuccinogenes Growing on Inulin

Sridhar

Eiteman

Wiegel

2000

Appl Environ Microbiol

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“…SDS-PAGE of the purified enzyme revealed one subunit with relative molecular mass equal to 73 kDa, which suggests an (X2 subunit stoichiometry for the native enzyme (Fig. 2) (29). A Hill plot of the data resulted in a Hill coefficient of 1.58 ± 0.12, suggesting two interacting substrate sites (Fig.…”

Section: Methodsmentioning

confidence: 89%

“…A Hill plot of the data resulted in a Hill coefficient of 1.58 ± 0.12, suggesting two interacting substrate sites (Fig. 3) (29).…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii

Jetten¹,

Stams²,

Zehnder³

1989

J Bacteriol

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In Methanothrix soehngenu, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, inting that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngeniu estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular ma-of 73 kDa in an a2 oligomeric structure. The terminal step in the breakdown of organic polymers under methanogenic conditions is the conversion of H2/CO2 or acetate by methanogens (9). The most abundant methanogenic substrate under these conditions is acetate (9, 33).

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“…Phosphotransacetylase and acetate kinase have been isolated and biochemically characterized from microorganisms belonging to the Bacteria domain (7,11,16,24,25,32,34,36,37,38,45) and from Methanosarcina thermophila in the Archaea domain (1,21). Despite the importance of these enzymes in anaerobic carbon cycling and in the metabolism of both aerobic and anaerobic microbes, few molecular genetic approaches have been taken in the study of these two enzymes.…”

mentioning

confidence: 99%

Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila

Latimer

Ferry

1993

J Bacteriol

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The genes for the acetate-activating enzymes, acetate kinase and phosphotransacetylase (ack and pta), from Methanosarcina thermophila TM-1 were cloned and sequenced. Both genes are present in only one copy per genome, with the pta gene adjacent to and upstream of the ack gene. Consensus archaeal promoter sequences are found upstream of the pta coding region. The pta and ack genes encode predicted polypeptides with molecular masses of 35,198 and 44,482 Da, respectively. A hydropathy plot of the deduced phosphotransacetylase sequence indicates that it is a hydrophobic polypeptide; however, no membrane-spanning domains are evident. Comparison of the amino acid sequences deduced from the M. thermophila and Escherichia coli ack genes indicate similar subunit molecular weights and 44% identity (60% similarity). The comparison also revealed the presence of several conserved arginine, cysteine, and glutamic acid residues. Arginine, cysteine, and glutamic acid residues have previously been implicated at or near the active site of the E. coli acetate kinase. The pta and ack genes were hyperexpressed in E. coli, and the overproduced enzymes were purified to homogeneity with specific activities higher than those of the enzymes previously purified from M. thermophila. The overproduced phosphotransacetylase and acetate kinase migrated at molecular masses of 37,000 and 42,000 Da, respectively. The activity of the acetate kinase is optimal at 65°C and is protected from thermal inactivation by ATP. Diethylpyrocarbonate and phenylglyoxal inhibited acetate kinase activity in a manner consistent with the presence of histidine and arginine residues at or near the active site; however, the thiol-directed reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide were ineffective.The metabolism of acetate, whether through the production or consumption of the principal intermediate acetyl coenzyme A (acetyl-CoA), is of major importance in the physiology of both procaryotes (Bacteria and Archaea domains) and eucaryotes (Eucarya domain). Acetate is an end product in the energy-yielding metabolism of most facultatively and strictly anaerobic microbes. Phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) are synthesized by these anaerobes to catalyze the conversion of acetyl-CoA and ADP to acetate and ATP,

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Purification and properties of acetate kinase from Clostridium thermoaceticum

Cited by 66 publications

References 25 publications

Elucidation of Enzymes in Fermentation Pathways Used by Clostridium thermosuccinogenes Growing on Inulin

Elucidation of Enzymes in Fermentation Pathways Used by Clostridium thermosuccinogenes Growing on Inulin

Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii

Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila

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