Literature DB >> 10074080

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

A K Bock1, J Glasemacher, R Schmidt, P Schönheit.   

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 (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.

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Year:  1999        PMID: 10074080      PMCID: PMC93586     

Source DB:  PubMed          Journal:  J Bacteriol        ISSN: 0021-9193            Impact factor:   3.490


  39 in total

Review 1.  Biochemistry of acetate catabolism in anaerobic chemotrophic bacteria.

Authors:  R K Thauer; D Möller-Zinkhan; A M Spormann
Journal:  Annu Rev Microbiol       Date:  1989       Impact factor: 15.500

2.  Purification and properties of phosphotransacetylase from Veillonella alcalescens.

Authors:  H R Whiteley; R A Pelroy
Journal:  J Biol Chem       Date:  1972-03-25       Impact factor: 5.157

3.  Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

Authors:  U K Laemmli
Journal:  Nature       Date:  1970-08-15       Impact factor: 49.962

4.  Purification and properties of phosphotransacetylase from Lactobacillus fermenti.

Authors:  T Nojiri; F Tanaka; I Nakayama
Journal:  J Biochem       Date:  1971-04       Impact factor: 3.387

5.  Acetate kinase from Veillonella alcalescens. Regulation of enzyme activity by succinate and substrates.

Authors:  C M Bowman; R O Valdez; J S Nishimura
Journal:  J Biol Chem       Date:  1976-05-25       Impact factor: 5.157

6.  Purification and properties of an acetate kinase from Rhodopseudomonas palustris.

Authors:  H Vigenschow; H M Schwarm; K Knobloch
Journal:  Biol Chem Hoppe Seyler       Date:  1986-09

7.  Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824.

Authors:  Z L Boynton; G N Bennett; F B Rudolph
Journal:  Appl Environ Microbiol       Date:  1996-08       Impact factor: 4.792

8.  Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis.

Authors:  D J Aceti; J G Ferry
Journal:  J Biol Chem       Date:  1988-10-25       Impact factor: 5.157

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

Authors:  L L Lundie; J G Ferry
Journal:  J Biol Chem       Date:  1989-11-05       Impact factor: 5.157

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

Authors:  H L Drake; S I Hu; H G Wood
Journal:  J Biol Chem       Date:  1981-11-10       Impact factor: 5.157

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  21 in total

1.  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.

Authors:  M Musfeldt; M Selig; P Schönheit
Journal:  J Bacteriol       Date:  1999-09       Impact factor: 3.490

Review 2.  Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

Authors:  C Vieille; G J Zeikus
Journal:  Microbiol Mol Biol Rev       Date:  2001-03       Impact factor: 11.056

3.  Structure of NDP-forming Acetyl-CoA synthetase ACD1 reveals a large rearrangement for phosphoryl transfer.

Authors:  Renato H-J Weiße; Annette Faust; Marcel Schmidt; Peter Schönheit; Axel J Scheidig
Journal:  Proc Natl Acad Sci U S A       Date:  2016-01-19       Impact factor: 11.205

4.  A continuous assay of acetate kinase activity: measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate.

Authors:  Soma Mukhopadhyay; Miriam S Hasson; David A Sanders
Journal:  Bioorg Chem       Date:  2008-02-21       Impact factor: 5.275

5.  Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073.

Authors:  Yuki Iwasaki; Akihisa Kita; Koichiro Yoshida; Takahisa Tajima; Shinichi Yano; Tomohiro Shou; Masahiro Saito; Junichi Kato; Katsuji Murakami; Yutaka Nakashimada
Journal:  Appl Environ Microbiol       Date:  2017-03-31       Impact factor: 4.792

Review 6.  Carbohydrate metabolism in Archaea: current insights into unusual enzymes and pathways and their regulation.

Authors:  Christopher Bräsen; Dominik Esser; Bernadette Rauch; Bettina Siebers
Journal:  Microbiol Mol Biol Rev       Date:  2014-03       Impact factor: 11.056

7.  Crystal structures of a phosphotransacetylase from Bacillus subtilis and its complex with acetyl phosphate.

Authors:  Qian Steven Xu; Jarmila Jancarik; Yun Lou; Kate Kuznetsova; Alexander F Yakunin; Hisao Yokota; Paul Adams; Rosalind Kim; Sung-Hou Kim
Journal:  J Struct Funct Genomics       Date:  2005-11-09

8.  Novel type of ADP-forming acetyl coenzyme A synthetase in hyperthermophilic archaea: heterologous expression and characterization of isoenzymes from the sulfate reducer Archaeoglobus fulgidus and the methanogen Methanococcus jannaschii.

Authors:  Meike Musfeldt; Peter Schönheit
Journal:  J Bacteriol       Date:  2002-02       Impact factor: 3.490

Review 9.  Magnesium magnetic isotope effects in microbiology.

Authors:  Ulyana G Letuta
Journal:  Arch Microbiol       Date:  2021-02-21       Impact factor: 2.552

10.  Regulation of acetate kinase isozymes and its importance for mixed-acid fermentation in Lactococcus lactis.

Authors:  Pranav Puri; Anisha Goel; Agnieszka Bochynska; Bert Poolman
Journal:  J Bacteriol       Date:  2014-01-24       Impact factor: 3.490

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