Purification and Characterization of Two Extremely Thermostable Enzymes, Phosphate Acetyltransferase and Acetate Kinase, from the Hyperthermophilic Eubacterium Thermotoga maritima

Abstract

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 (α4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases fromClostridium 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 μM, respectively; the apparentV max values (at 55°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°C and was not inactivated by heat upon incubation at 80°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 (α2) 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 V max values (at 50°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°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°C for 3 h. The temperature optimum at 90°C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

A continuous assay of acetate kinase activity: Measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate

“…A lower K m of the Thermotoga maritima acetate kinase for acetate was measured than that determined by earlier assay methods. The Km of Thermotoga maritima acetate kinase was previously determined to be 40mM when measured at 55° C (11). The lower Km value measured here is more consistent with the concentrations of acetate to which cells are exposed and the internal acetyl phosphate concentrations that have been measured experimentally (12).…”

Cloning of the phosphotransacetylase gene from Lactobacillus sanfranciscensis and characterization of its gene product

“…The K m values of the L. sanfranciscensis PTA were within the ranges reported for other organisms (NOJIRI et al 1971, REINSCHEID et al 1999, BOCK et al 1999. Generally, PTAs have higher values for acetyl-CoA and phosphate than for CoA and acetyl phosphate, respectively.…”

Hyperthermophilic aldolases as biocatalyst for C–C bond formation: rhamnulose 1-phosphate aldolase from Thermotoga maritima

“…Usually, most enzymes characterized from hyperthermophiles have a maximum or optimal temperature near the microorganism's optimum growth temperature. However, T. maritima seems to behave differently because, asides from the aldolase described in this work, many of the enzymes isolated from this organism exhibit a similar performance with optimum temperatures around 95 °C (Bock et al 1999;Song et al 2008;Drzewiecki et al 2010;Park et al 2010;Huang et al 2012). This fact could explain the capacity of this eubacteria to grow at temperatures above 90 °C (Huber et al 1986).…”