The cytoplasmic cyclophilin from <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> interacts with phosphate acetyltransferase isoforms enhancing their in vitro activity

Cyclophilins belong to the peptidyl-prolyl cis/trans isomerase family of enzymes (EC 5.2.1.8), which accelerate protein folding by catalysing the cis/trans isomerisation of proline imidic peptide bonds. In the present study, by a combination of bioinformatics methods, we identify phosphate acetyltransferase isoforms, AvPTA-1 and AvPTA-2, as potential interacting partners of AvPPIB, the cytoplasmic cyclophilin from Azotobacter vinelandii, and demonstrate their physical interaction by co-expression studies. A decrease in AvPPIB PPIase activity, in the presence of AvPTA-1 or AvPTA-2, further confirms each interaction. Phosphate acetyltransferases (EC 2.3.1.8) catalyse the reversible transfer of the acetyl group from acetyl-P to CoA, forming acetyl-CoA and inorganic phosphate. We examined the effect of AvPPIB on the enzymatic activity of both phosphate acetyltransferase isoforms, and noticed an enhancement of the activity, as well as an alteration of the K _m of each isoform, for the reaction substrates, indicating a possible function of AvPPIB in phosphate acetyltransferase activity modulation. Although PPIase activity seems not to be essential for these interactions, since AvPPIB_F99A active site mutant still interacts with both isoforms, it is responsible for the observed phosphate acetyltransferase activity enhancement as AvPPIB_F99A enhanced to a significantly lower extent the phosphate acetyltransferase activity of both isoforms compared with AvPPIB.     

Different mechanisms of acetate activation in Desulfurella acetivorans and Desulfuromonas acetoxidans

Desulfurella acetivorans and Desulfuromonas acetoxidans are both acetate oxidizing sulfur reducing eubacteria. The two organisms differ in G+C content of DNA (31.4% versus 50–52%) and in growth temperature optimum (55°C versus 30°C) and in that D. acetivorans does not contain cytochromes. Both organisms are shown to be similar in that they metabolize acetate via the citric acid cycle rather than via the carbon monoxide dehydrogenase pathway. They were found to differ, however, in the mechanism of acetate activation and of succinate formation. In D. acetoxidans acetyl-CoA and succinate are formed from acetate and succinyl-CoA involving only one enzyme, succinyl-CoA: acetate CoA-transferase. In D. acetivorans acetyl-CoA is generated from acetate via acetyl phosphate involving acetate kinase and phosphate acetyltransferase; succinate is formed from succinyl-CoA via succinyl-CoA synthetase. Both sulfur reducers were found to contain menaquinone.Abbreviations HPLC high performance liquid chromatography - acetyl-P acetyl phosphate     

Acetyl-CoA synthetase (ADP forming) in archaea,a novel enzyme involved in acetate formation and ATP synthesis

The anaerobic hyperthermophilic archaea Desulfurococcus amylolyticus, Hyperthermus butylicus, Thermococcus celer, Pyrococcus woesei, the hyperthermophilic bacteria Thermotoga maritima and Clostridium thermohydrosulfuricum and the aerobic mesophilic archaeon Halobacterium saccharovorum were grown either on complex media, on sugars or on pyruvate as carbon and energy sources. During growth acetate was formed as fermentation product by all organisms. The enzymes involved in acetyl-CoA formation from pyruvate and in acetate formation from acetyl-CoA were investigated:

1. Cell extracts of all species, both archaea and bacteria, catalyzed the coenzyme A-dependent oxidative decarboxylation of pyruvate with viologen dyes or with Clostridium pasteurianum ferredoxin as electron acceptors indicating a pyruvate: ferredoxin oxidoreductase to be operative in acetyl-CoA formation from pyruvate.
2. Cell extracts of all archaeal species, both hyperthermophiles (D. amylolyticus, H. butylicus, T. celer, P. woesei) and the mesophile H. saccharovorum, contained an acetyl-CoA synthetase (ADP forming), which catalyzes both acetate formation from acetyl-CoA and ATP synthesis from ADP and phosphate (P_i): Acetyl-CoA+ADP+P_i?Acetate + ATP+CoA. Phosphate acetyltransferase and acetate kinase could not be detected.
3. Cell extracts of the hyperthermophilic (eu)bacteria T. maritima and C. thermohydrosulfuricum contained phosphate acetyltransferase and acetate kinase rather than acetyl-CoA synthetase (ADP forming).

These data indicate that acetyl-CoA synthetase (ADP forming) represents a typical archaeal property rather than an enzyme specific for hyperthermophiles. It is proposed that in all acetate forming archaea the formation of acetate and of ATP from acetyl-CoA, ADP and P_i are catalyzed by acetyl-CoA synthetase (ADP forming), whereas in all acetate forming (eu)bacteria these reactions are catalyzed by two enzymes, phosphate acetyltransferase and acetate kinase.     

Production of isoamyl acetate in<Emphasis Type="Italic"> ackA-pta</Emphasis> and/or<Emphasis Type="Italic"> ldh</Emphasis> mutants of<Emphasis Type="Italic"> Escherichia coli</Emphasis> with overexpression of yeast<Emphasis Type="Italic"> ATF2</Emphasis>

The gene coding for alcohol acetyltransferase (ATF2), which catalyzes the esterification of isoamyl alcohol and acetyl coenzyme A (acetyl-CoA), was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli. This genetically engineered strain of E. coli produced the ester isoamyl acetate when isoamyl alcohol was added externally to the cell culture medium. Various competing pathways at the acetyl-CoA node were inactivated to increase the intracellular acetyl-CoA pool and divert more carbon flux to the ester synthesis pathway. Several strains with deletions in the ackA-pta and/or ldh pathways and bearing the ATF2 on a high-copy-number plasmid were constructed and studied. Compared to the wild-type, ackA-pta and nuo mutants produced higher amounts of ester and an ackA-pta-ldh-nuo mutant lower amounts. Isoamyl acetate production correlated well with intracellular coenzyme A (CoA) and acetyl-CoA levels. The ackA-pta-nuo mutant had the highest intracellular CoA/acetyl-CoA level and hence produced the highest amount of ester (1.75 mM) during the growth phase under oxic conditions and during the production phase under anoxic conditions.     

Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering

The overexpression of acetyl-CoA (CoA) synthetase (ACS) in Escherichia coli showed significant reduction in acetate during glucose fermentation. It also greatly enhanced acetate assimilation when acetate was used as a carbon source. These features are ideal for applications in metabolic engineering. ACS overexpression can be strategically applied to reduce acetate byproduct, recover wasted carbon, and redirect carbon flux toward more favorable pathways. The native acs gene was cloned and overexpressed in E. coli. Studies showed significant effects on acetate production and assimilation in cultures grown in minimal and complex media with glucose or acetate as the carbon source.     

A Study of the Mechanism of Acetate Assimilation in Purple Nonsulfur Bacteria Lacking the Glyoxylate Shunt: Enzymes of the Citramalate Cycle in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The mechanism of acetate assimilation by the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shunt, has been studied. In a previous work, proceeding from data on acetate assimilation by Rba. sphaeroides cell suspensions, a suggestion was made regarding the operation, in this bacterium, of the citramalate cycle. This cycle was earlier found in Rhodospirillum rubrum in the form of an anaplerotic reaction sequence that operates during growth on acetate instead of the glyoxylate shunt, which is not present in the latter bacterium. The present work considers the enzymes responsible for acetate assimilation in Rba. sphaeroides. It is shown that this bacterium possesses the key enzymes of the citramalate cycle: citramalate synthase, which catalyzes condensation of acetyl-CoA and pyruvate and, as a result, forms citramalate, and 3-methylmalyl-CoA lyase, which catalyzes the cleavage of 3-methylmalyl-CoA to glyoxylate and propionyl-CoA. The regeneration of pyruvate, which is the acetyl-CoA acceptor in the citramalate cycle, involves propionyl- CoA and occurs via the following reaction sequence: propionyl-CoA (+CO₂) å methylmalonyl-CoA å succinyl-CoA å succinate å fumarate malate å oxaloacetate (−CO₂) å phosphoenolpyruvate å pyruvate. The independence of the cell growth and the acetate assimilation of CO₂ is due to the accumulation of CO₂/HCO ₃ ⁻ (released during acetate assimilation) in cells to a level sufficient for the effective operation of propionyl-CoA carboxylase.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 319–328.Original Russian Text Copyright © 2005 by Filatova, Berg, Krasil’nikova, Ivanovsky.     

Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PP_i-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PP_i were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PP_i displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.     

Modulating aroma compounds during wine fermentation by manipulating carnitine acetyltransferases in Saccharomyces cerevisiae

The wine yeast Saccharomyces cerevisiae is central in the production of aroma compounds during fermentation. Some of the most important yeast-derived aroma compounds produced are esters. The esters ethyl acetate and isoamyl acetate are formed from alcohols and acetyl-CoA in a reaction catalysed by alcohol acetyltransferases. The pool of acetyl-CoA available in yeast cells could play a key role in the development of ester aromas. Carnitine acetyltransferases catalyse the reversible reaction between carnitine and acetyl-CoA to form acetylcarnitine and free CoA. This reaction is important in transferring activated acetyl groups to the mitochondria and in regulating the acetyl-CoA/CoA pools within the cell. We investigated the effect of overexpressing CAT2, which encodes the major mitochondrial and peroxisomal carnitine acetyltransferase, on the formation of esters and other flavour compounds during fermentation. We also overexpressed a modified CAT2 that results in a protein that localizes to the cytosol. In general, the overexpression of both forms of CAT2 resulted in a reduction in ester concentrations, especially in ethyl acetate and isoamyl acetate. We hypothesize that overproduction of Cat2p favours the formation of acetylcarnitine and CoA and therefore limits the precursor for ester production. Carnitine acetyltransferase expression could potentially to be used successfully in order to modulate wine flavour.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

Mechanisms of acetate formation and acetate activation in halophilic archaea

The halophilic archaea Halococcus (Hc.) saccharolyticus, Haloferax (Hf.) volcanii, and Halorubrum (Hr.) saccharovorum were found to generate acetate during growth on glucose and to utilize acetate as a growth substrate. The mechanisms of acetate formation from acetyl-CoA and of acetate activation to acetyl-CoA were studied. Hc. saccharolyticus, exponentially growing on complex medium with glucose, formed acetate and contained ADP-forming acetyl-CoA synthetase (ADP-ACS) rather than acetate kinase and phosphate acetyltransferase or AMP-forming acetyl-CoA synthetase. In the stationary phase, the excreted acetate was completely consumed, and cells contained AMP-forming acetyl-CoA synthetase (AMP-ACS) and a significantly reduced ADP-ACS activity. Hc. saccharolyticus, grown on acetate as carbon and energy source, contained only AMP-ACS rather than ADP-ACS or acetate kinase. Cell suspensions of Hc. saccharolyticus metabolized acetate only when they contained AMP-ACS activity, i.e., when they were obtained after growth on acetate or from the stationary phase after growth on glucose. Suspensions of exponential glucose-grown cells, containing only ADP-ACS but not AMP-ACS, did not consume acetate. Similar results were obtained for the phylogenetic distantly related halophilic archaea Hf. volcanii and Hf. saccharovorum. We conclude that, in halophilic archaea, the formation of acetate from acetyl-CoA is catalyzed by ADP-ACS, whereas the activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS.Abbreviations. Hc. Halococcus - Hf. Haloferax - Hr. Halorubrum - Hb. Halobacterium An erratum to this article can be found at     

Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli

We investigated the relationship between Escherichia coli flagellar expression and the regulation of acetyl phosphate synthesis and degradation. Using cells either wild type for acetyl phosphate metabolism or defective for phosphotransacetylase or acetate kinase, or both, we measured flagellar expression and the intracellular concentration of acetyl phosphate relative to growth phase and temperature. Under the conditions tested, we found that elevated levels of acetyl phosphate corresponded to inhibition of flagellar synthesis. To extend these observations, we measured the intracellular concentration of acetyl-CoA, the level of expression from the pta and ackA promoters, and the activities of phosphotransacetylase and acetate kinase derived from cell lysates. Relative to increasing culture density, acetyl-CoA levels and expression from both the pta and ackA promoters decreased. Relative to Increasing temperature, expression from the ackA promoter decreased and phosphotransacetylase activity increased. In contrast, temperature had little or no effect on either acetate kinase activity or expression from the pta promoter. We propose that cells regulate intracellular acetyl phosphate concentrations relative to growth phase and temperature by modulating the availability of acetyl-CoA, the expression of ackA, and the activity of phosphotransacetylase.     

Biosynthesis of acetyl-coenzyme A in the electric organ of Torpedo marmorata in relation to acetylcholine metabolism.

Formation of acetyl-CoA through acetyl-CoA synthetase (forward reaction) and through choline acyltransferase (backward reaction) was investigated in tissue extract from the electric organ of Torpedo marmorata. When the tissue extract was submitted to gel filtration on Sephadex G-25, the formation of acetyl-CoA by acetyl-CoA synthetase appeared fully dependent on ATP and CoA and partially dependent on acetate (an endogenous supply of acetate is discussed). Choline acetyltransferase was a potent source of acetyl-CoA, only requiring acetylcholine and CoA, and was much more efficient than acetyl-CoA synthetase for concentrations of acetylcholine likely to be present in nerve endings.     

Regulation of Acetate Metabolism in a Strain of Acinetobacter sp. Growing on Ethanol

Ethanol metabolism in Acinetobacter sp. is shown to be limited by the rate of acetate assimilation, a reaction catalyzed by acetyl-CoA synthetase (EC 6.2.1.1). Effects of ions (sodium, potassium, and magnesium), by-products of ethanol and acetaldehyde oxidation (NADH and NADPH), and pantothenic acid on this enzyme are studied (sodium, NADH, and NADPH inhibit acetyl-CoA synthetase; pantothenic acid, potassium, and magnesium act as enzyme activators). Conditions of culturing were developed under which ethanol, acetaldehyde, and acetate in Acinetobacter cells were oxidized at the same rates, producing a threefold increase in the activity of acetyl-CoA synthetase in the cell-free extract. The results of studies of acetyl-CoA synthetase regulation in a mutant strain of Acinetobacter sp., which is incapable of forming exopolysaccharides, provide a basis for refining the technology of ethapolan production involving the use of C₂ substrates.     

Transcriptional and biochemical characterization of two <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> FKBP family members

Peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), a class of enzymes that catalyse the rate-limiting step of the cis/trans isomerization in protein folding, are divided into three structurally unrelated families: cyclophilins, FK506-binding proteins (FKBPs), and parvulins. Two recombinant FKBPs from the soil nitrogen-fixing bacterium Azotobacter vinelandii, designated as AvfkbX and AvfkbB, have been purified and their peptidyl-prolyl cis/trans isomerase activity against Suc-Ala-Xaa-Pro-Phe-pNA synthetic peptides characterised. The substrate specificity of both enzymes is typical for bacterial FKBPs, with Suc-Ala-Phe-Pro-Phe-pNA being the most rapidly catalysed substrate by AvfkbX and Suc-Ala-Leu-Pro-Phe-pNA by AvfkbB. Both FKBPs display chaperone activity as well in the citrate synthase thermal aggregation assay. Furthermore, using real-time RT-qPCR, we demonstrated that both genes were expressed during the exponential growth phase on glucose minimal medium, while their expression declined dramatically during the stationary growth phase as well as when the growth medium was supplied exogenously with ammonium.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM OX BRAIN STRIATE NUCLEI

Abstract—

• 1 Choline acetyltransferase was purified from ox brain striate nuclei by an extraction step at pH 5, cation-exchange chromatography, fractional precipitation with ammonium sulphate, and chromatography on Sephadex G-200. The enzyme was obtained free of deacylases and cholinesterases, at specific activities of 01-0-3 μmol acetylcholine formed per min per mg protein.
• 2 The enzyme was found to be a stable and relatively basic protein, with a molecular weight of 65,000.
• 3 In the catalysed reactions, , k₁, was about four times k₂, and the equilibrium constant was approximately 40. For the forward reaction, the Michaelis constant for each substrate was independent of the concentration of the other (choline = 0-75 mM; acetyl-CoA = 10 μM), whereas in the back reaction one substrate increased the affinity for the other (acetylcholine = 0-75-5 MM; CoA = 25-150 μM).
• 4 CoA inhibited acetylcholine synthesis by competing with acetyl-CoA (K₁, = 16 μM). Acetylcholine slightly inhibited the forward reaction (e.g. 45 per cent in 200 mM) without competing with choline or acetyl-CoA. These data indicate an ordered reaction mechanism; acetyl-CoA probably always binds before choline.

     

Proteomic investigation of the impact of oxygen on the protein profiles of hyaluronic acid‐producing Streptococcus zooepidemicus

Hyaluronic acid (HA) is a linear and negatively charged polysaccharide regularly used in medicine and cosmetics. Recently Streptococcus zooepidemicus has been exploited in the fermentation industry to produce HA. Many studies showed that higher amounts of HA were produced under aerobic condition compared to anaerobic conditions. To explore the effect of oxygen on the HA synthesis in S. zooepidemicus, 2‐DE was used to compare the proteomes of aerobically and anaerobically fermented bacteria to identify proteins, which might be associated with the influence of oxygen on the HA synthesis. Totally nine pairs of 2‐DE gels collected from three batches were compared and nine overexpressed proteins were observed in aerobically fermented bacteria. These proteins were identified by LC/tandem MS as dihydrolipoamide dehydrogenase, UDP‐acetyl‐glucosamine pyrophosphoylase, dihydrolipoamide‐S‐acetyltransferase and acetoin dehydrogenase α and β chains, respectively. These upregulated proteins were involved in acetoin dissimilation, the central carbon metabolism and the HA anabolic pathway, implicating that oxygen might augment the expression of genes that are involved in central energy metabolism, acetoin reutilization and HA biosynthesis to enhance the amount of acetyl‐CoA as such that more acetyl‐CoA can be diverged from the central carbon metabolism to replenish acetyl‐CoA for the HA synthesis.     

Metabolism of dimethylsulphoniopropionate by Ruegeria pomeroyi DSS‐3

Ruegeria pomeroyi DSS‐3 possesses two general pathways for metabolism of dimethylsulphoniopropionate (DMSP), an osmolyte of algae and abundant carbon source for marine bacteria. In the DMSP cleavage pathway, acrylate is transformed into acryloyl‐CoA by propionate‐CoA ligase (SPO2934) and other unidentified acyl‐CoA ligases. Acryloyl‐CoA is then reduced to propionyl‐CoA by AcuI or SPO1914. Acryloyl‐CoA is also rapidly hydrated to 3‐hydroxypropionyl‐CoA by acryloyl‐CoA hydratase (SPO0147). A SPO1914 mutant was unable to grow on acrylate as the sole carbon source, supporting its role in this pathway. Similarly, growth on methylmercaptopropionate, the first intermediate of the DMSP demethylation pathway, was severely inhibited by a mutation in the gene encoding crotonyl‐CoA carboxylase/reductase, demonstrating that acetate produced by this pathway was metabolized by the ethylmalonyl‐CoA pathway. Amino acids and nucleosides from cells grown on ¹³C‐enriched DMSP possessed labelling patterns that were consistent with carbon from DMSP being metabolized by both the ethylmalonyl‐CoA and acrylate pathways as well as a role for pyruvate dehydrogenase. This latter conclusion was supported by the phenotype of a pdh mutant, which grew poorly on electron‐rich substrates. Additionally, label from [¹³C‐methyl] DMSP only appeared in carbons derived from methyl‐tetrahydrofolate, and there was no evidence for a serine cycle of C‐1 assimilation.     

Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima

Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/<-- acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 microM, respectively; the apparent Vmax values (at 55 degrees C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90 degrees C and was not inactivated by heat upon incubation at 80 degrees C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (alpha2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP -->/<-- acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.