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Archives of Microbiology

, Volume 156, Issue 6, pp 517–524 | Cite as

N5,N10-Methenyltetrahydromethanopterin cyclohydrolase from the extreme thermophile Methanopyrus kandleri: increase of catalytic efficiency (kcat/KM) and thermostability in the presence of salts

  • J. Breitung
  • R. A. Schmitz
  • K. O. Stetter
  • R. K. Thauer
Original Papers
  • 112 Downloads

Abstract

The activity of purified N5,N10-methenyltetrahydromethanopterin cyclohydrolase from Methanopyrus kandleri was found to increase up to 200-fold when potassium phosphate was added in high concentrations (1.5 M) to the assay. A 200-fold stimulation was also observed with sodium phosphate (1 M) and sodium sulfate (1 M) whereas stimulation by potassium sulfate (0.8 M), ammonium sulfate (1.5 M), potassium chloride (2.5 M), and sodium chloride (2 M) was maximal 100-fold. A detailed kinetic analysis of the effect of potassium phosphate revealed that this salt exerted its stimulatory effect by decreasing the Km for N5,N10-methenyltetrahydromethanopterin from 2 mM to 40 μM and by increasing the Vmax from 2000 U/mg (kcat=1385 s-1) to 13300 U/mg (kcat=9200 s-1). Besides increasing the catalytic efficiency (kcat/Km) salts were found to protect the cyclohydrolase from heat inactivation. For maximal thermostability much lower concentrations (0.1 M) of salts were required than for maximal activity.

Key words

Methanogenic bacteria Archaebacteria Methanopyrus Hyperthermophiles Thermostability Tetrahydromethanopterin 

Abbreviations

H4MPT

tetrahydromethanopterin

\({\text{CH}} \equiv {\text{H}}_{\text{4}} {\text{MPT}}^{\text{ + }} \)

N5,N10-methenyl-H4MPT

CHO-H4MPT

N5-formyl-H4-MPT

CH2=H4MPT

N5,N10-methylene-H4MPT

CH3−H4-MPT

N5-methyl-H4MPT

MOPS

γ-N-morpholinopropane sulfonic acid

TRICINE

N-[Tris(hydroxymethyl)-methyl]glycine

1 U =

1 mol/min

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References

  1. Beelen P van, Stassen APM, Bosch JWG, Vogels GD, Guijt W, Haasnoot CAG (1984a) Elucidation of the structure of methanopterin, a coenzyme from Methanobacterium thermoautotrophicum, using two-dimensional nuclear-magnetic-resonance techniques. Eur J Biochem 138:563–571CrossRefGoogle Scholar
  2. Beelen P van, Labro JFA, Keltjens JT, Geerts WJ, Vogels GD, Laarhoven WH, Guijt W, Haasnoot CAG (1984b) Derivatives of methanopterin, a coenzyme involved in methanogenesis. Eur J Biochem 139:359–365CrossRefGoogle Scholar
  3. Beelen P van, Cock RM de, Guijt W, Haasnoot CAG, Vogels GD (1984c) Isolation and identification of 5,10-methenyl-5,6,7,8-tetrahydromethanopterin, a coenzyme involved in methanogenesis. FEMS Microbiol Lett 21:159–163CrossRefGoogle Scholar
  4. Bio-Rad Laboratories (1981) Instruction manual for Bio-Rad protein assay, Bio-Rad Laboratories, Richmond, Calif., USAGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  6. Breitung J, Thauer RK (1990) Formylmethanofuran: tetrahydromethanopterin formyltransferase from Methanosarcina barkeri: Identification of N 5-formyltetrahydromethanopterin as the product. FEBS Lett 275:226–230CrossRefGoogle Scholar
  7. Burggraf S, Stetter KO, Rouvière P, Woese CR (1991) Methanopyrus kandleri: An archaeal methanogen unrelated to all other known methanogens. System Appl Microbiol (in press)Google Scholar
  8. DiMarco AA, Donnelly MI, Wolfe RS (1986) Purification and properties of the 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanobacterium thermoautotrophicum. J Bacteriol 168:1372–1377CrossRefGoogle Scholar
  9. DiMarco AA, Bobik TA, Wolfe RS (1990) Unusual coenzymes of methanogenesis. Annu Rev Biochem 59:355–394CrossRefGoogle Scholar
  10. Donnelly MI, Escalante-Semerana JC, Rinehart KLJr, Wolfe RS (1985) Methenyl-tetrahydromethanopterin cyclohydrolase in cell extracts of Methanobacterium. Arch Biochem Biophys 242:430–439CrossRefGoogle Scholar
  11. Donnelly MI, Wolfe RS (1986) The role of formylmethanofuran: tetrahydromethanopterin formyltransferase in methanogenesis from carbon dioxide. J Biol Chem 261:16653–16659PubMedGoogle Scholar
  12. Görg A, Postel W, Günther S (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients (a review). Electrophoresis 9:531–546CrossRefGoogle Scholar
  13. Hensel R, König H (1988) Thermoadaptation of methanogenic bacteria by intracellular ion concentration. FEMS Microbiol Lett 49:75–79CrossRefGoogle Scholar
  14. Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ (1981) A gasliquid solid phase peptide and protein sequenator. J Biol Chem 256:7990–7997Google Scholar
  15. Huber R, Kurr M, Jannasch HW, Stetter KO (1989) A novel group of abyssal methanogenic archaebacteria (Methanopyrus) growing at 110° C. Nature 342:833–834CrossRefGoogle Scholar
  16. Jablonski PE, Ferry JG (1991) Purification and properties of methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila. J Bacteriol 173:2481–2487CrossRefGoogle Scholar
  17. Kanodia S, Roberts MF (1983) Methanophosphagen: unique cyclic pyrophosphate isolated from Methanobacterium thermoautotrophicum. Proc Natl Acad Sci USA 80:5217–5221CrossRefGoogle Scholar
  18. Keltjens JT, Caerteling GC, Drift C van der, Vogels GD (1986) Methanopterin and the intermediary steps of methanogenesis. System Appl Microbiol 7:370–375CrossRefGoogle Scholar
  19. Keltjens JT, Vogels GD (1988) Minireview: methanopterin and methanogenic bacteria. BioFactors 1:95–103PubMedGoogle Scholar
  20. Kurr M, Huber R, König H, Jannasch HW, Fricke H, Trincone A, Kristjansson JK, Stetter KO (1991) Methanopyrus kandleri, gen. and sp. nov. represents a novel group of hyperthermophilic methanogens, growing at 110° C. Arch Microbiol 156:239–247CrossRefGoogle Scholar
  21. Ma K, Linder D, Stetter KO, Thauer RK (1991a) Purification and properties of N 5,N 10-methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) from the extreme thermophile Methanopyrus kandleri. Arch Microbiol 155:593–600CrossRefGoogle Scholar
  22. Ma K, Zirngibl C, Linder D, Stetter KO, Thauer RK (1991b) N 5,N 10-Methylenetetrahydromethanopterin dehydrogenase (H2-forming) from Methanopyrus kandleri. Arch Microbiol 156:43–48CrossRefGoogle Scholar
  23. Perrin DD, Dempsey B (1974) Buffers for pH and metal ion control. Chapman and Hall Laboratory, LondonGoogle Scholar
  24. Rospert S, Breitung J, Ma K, Schwörer B, Zirngibl C, Thauer RK, Linder D, Huber R, Stetter KO (1991) Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri. Arch Microbiol 156:49–55CrossRefGoogle Scholar
  25. Rospert S, Linder D, Ellermann J, Thauer RK (1990) Two genetically distinct methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum strain Marburg and △ H. Eur J Biochem 194:871–877CrossRefGoogle Scholar
  26. Rudnick H, Hendrich S, Pilatus U, Blotevogel KH (1990) Phosphate accumulation and the occurrence of polyphosphates and cyclic 2,3-diphosphoglycerate in Methanosarcina frisia. Arch Microbiol 154:584–588CrossRefGoogle Scholar
  27. Schmitz RA, Linder D, Stetter KO, Thauer RK (1991) N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus. Arch Microbiol (in press)Google Scholar
  28. Schwörer B, Thauer RK (1991) Activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductas, and heterodisulfide reductase in methanogenic bacteria. Arch Microbiol 155:459–465CrossRefGoogle Scholar
  29. Seely RJ, Fahrney DE (1983) A novel diphospho-P,P′-diester from Methanobacterium thermoautotrophicum. J Biol Chem 258:10835–10838PubMedGoogle Scholar
  30. Te Brömmelstroet BW, Hensgens CMH, Geerts WJ, Keltjens JT, Drift C van der, Vogels GD (1990) Purification and properties of 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri. J Bacteriol 172:564–571CrossRefGoogle Scholar
  31. Thauer RK (1990) Energy metabolism of methanogenic bacteria. Biochim Biophys Acta 1018:256–259CrossRefGoogle Scholar
  32. Voet D, Voet JG (1990) Biochemistry. John Wiley & Sons, New York, p 338Google Scholar
  33. Woese CR, Kandler O, Wheelis ML (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 87:4576–4579CrossRefGoogle Scholar
  34. Zirngibl C, Hedderich R, Thauer RK (1990) N 5,N 10-Methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum has hydrogenase activity. FEBS Lett 261:112–116CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • J. Breitung
    • 1
  • R. A. Schmitz
    • 1
  • K. O. Stetter
    • 2
  • R. K. Thauer
    • 1
  1. 1.Laboratorium für Mikrobiologie, Fachbereich BiologiePhilipps-Universität MarburgMarburg/LahnGermany
  2. 2.Lehistuhl für MikrobiologieUniversität RegensburgRegensburgGermany

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