Acetate kinase and phosphotransacetylase.

Most of the methane produced in nature derives from the methyl group of acetate, the major end product of anaerobes decomposing complex plant material. The acetate is derived from the metabolic intermediate acetyl-CoA via the combined activities of phosphotransacetylase and acetate kinase. In Methanosarcina species, the enzymes function in the reverse direction to activate acetate to acetyl-CoA prior to cleavage into a methyl and carbonyl group of which the latter is oxidized providing electrons for reduction of the former to methane. Thus, phosphotransacetylase and acetate kinase have a central role in the conversion of complex organic matter to methane by anaerobic microbial food chains. Both enzymes have been purified from Methanosarcina thermophila and characterized. Both enzymes from M. thermophila have also been produced in Escherichia coli permitting crystal structures and amino acid variants, the kinetic and biochemical studies of which have lead to proposals for catalytic mechanisms. The high identity of both enzymes to paralogs in the domain Bacteria suggests ancient origins and common mechanisms.

Expression, purification, crystallization and preliminary X-ray analysis of phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase (Pta) from the anaerobic archaeon Methanosarcina thermophila has been heterologously expressed in a soluble form which facilitated crystallization using the hanging-drop vapor-diffusion method with ammonium sulfate as a precipitant. This is the first report of the crystallization of any Pta. While the M. thermophila Pta has high sequence identity to Ptas from other organisms, it has no homology to any previously crystallized proteins. The protein crystallized in space group I4(1), with unit-cell parameters a = b = 114.8, c = 127.8 A, alpha = beta = gamma = 90 degrees. The crystals diffracted to 2.5 A resolution using Cu Kalpha radiation. The enzyme had previously been reported to exist as a monomer; however, the self-rotation function showed the presence of a non-crystallographic symmetry axis at psi = 90, phi = 90, kappa = 180 degrees, suggesting oligomerization. Dynamic light-scattering analysis supported a dimeric state for Pta in solution.

Exploitation of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid).

Active butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) were purified in three steps: ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on Matrex Red A from recombinant Escherichia coli K2006 (pJC7). They were then successfully exploited for in vitro synthesis of 3-hydroxybutyryl-CoA (3HBCoA), 4-hydroxybutyryl-CoA (4HBCoA), 4-hydroxyvaleryl-CoA (4HVCoA) and poly(hydroxyalkanoic acid) (PHA). In addition, the ability of the PHA synthase of Chromatium vinosum, PhaEC(Cv), to use these CoA thioesters was evaluated. Combination of Buk and Ptb with PhaEC(Cv) established a new system for in vitro synthesis of poly(3-hydroxybutyric acid) [poly(3HB)]. In this system, 3-hydroxybutyric acid was converted to 3HBCoA by Buk and Ptb at the expense of ATP. Formation of 3HBCoA was further driven by the polymerization of 3HBCoA molecules to poly(3HB) by PHA synthase, and the released CoA was recycled by Ptb. This system therefore also ensured the regeneration of CoA. With ATP as the energy supply, which was hydrolyzed to ADP and phosphate, 2.6 mg poly(3HB) was obtained from a 1-ml reaction mixture containing 7.6 mg 3-hydroxybutyrate at the beginning. Studies showed that Ptb and PHA synthase were the rate-limiting steps in this system, and initial CoA concentrations ranging from 1 to 7 mM did not inhibit poly(3HB) synthesis. Synthesis of various polyesters of 3HB and 4HB with this system was also tested, and copolyesters containing 4HB of 1-46 mol % were obtained.

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.

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

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 polypeptides; 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 degrees 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.

Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase.

Phosphotransacetylase (EC 2.3.1.8) was purified 83-fold to a specific activity of 2.5 mmol of acetyl-CoA synthesized per min/mg of protein from Methanosarcina thermophila cultivated on acetate. This rate was 10-fold greater than the rate of acetyl phosphate synthesis. The native enzyme (Mr 42,000-52,000) was a monomer and was not integral to the membrane. Activity was optimum at pH 7.0, and 35-45 degrees C. The enzyme was stable to air and to temperatures up to 70 degrees C, but was inactivated at higher temperatures. Phosphate and sulfate partially protected against heat inactivation. Potassium or ammonium ion concentrations above 10 mM were required for maximum activity of the purified enzyme; the intracellular potassium concentration of M. thermophila approximated 175 mM. Sodium, phosphate, sulfate, and arsenate ions were inhibitory to enzyme activity. Western blots of cell extracts showed that phosphotransacetylase was synthesized in higher quantity in acetate-grown cells than in methanol-grown cells.

Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis.

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.

Purification and characterization of a phosphotransacetylase from Rhodopseudomonas palustris.

A phosphotransacetylase was purified to apparent homogeneity from photolithoautotrophically grown Rhodopseudomonas palustris by liquid chromatography methods. A 400-fold increase in specific activity could be achieved. The enzyme was characterized by a relative molecular mass of 54,500, an isoelectric point of 6.3 and the absence of dissociable subunits. The enzyme appeared very labile at elevated temperatures or in diluted solutions. The stability could be increased distinctly in case sulfate or ammonium ions were added to the enzyme solution. The activity also was influenced by inorganic salts. Potassium and ammonium ions activated the enzymatic reaction. Sulfate ions revealed an inhibitory influence. A strong substrate inhibition was found with coenzyme A as substrate. The Arrhenius plot revealed a discontinuity at 15 degrees C which most likely corresponds to a conformational change of the enzyme protein.

Purification of five components from Clostridium thermoaceticum which catalyze synthesis of acetate from pyruvate and methyltetrahydrofolate. Properties of phosphotransacetylase.

A five-component enzyme system which catalyzes synthesis of acetylphosphate from methyltetrahydrofolate (CH3THF) plus pyruvate has been purified from the homoacetate-fermenting bacterium, Clostridium thermoaceticum. One of the components was identified as the low potential electron carrier, ferredoxin, and the other 4 protein components have been designated F1, F2, F3, and F4. F1, F2, and F4 have been purified to homogeneity and, as estimated by gel filtration, have native molecular weights of 88,100, 58,900, and 255,000, respectively, while the subunit molecular weights obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 20,000, 25,500, and 120,000, respectively. F3 contains 3 to 4 protein bands and has not been characterized with respect to molecular weights. Acetylphosphate synthesis by the purified system is optimal at pH 6.0 and 65 degrees C and requires ATP, CoA, and, to a lesser extent, thiamin pyrophosphate and Fe2+. S-Adenosylmethionine is not required. The F1 component has been identified as phosphotransacetylase and in its absence, the product is acetyl-CoA. Some properties of the phosphotransacetylase are presented. A scheme is given indicating present views of the functions of the individual components.