Molecular and General Genetics MGG

, Volume 242, Issue 2, pp 163–168 | Cite as

Cloning, sequencing and expression of the gene encoding 2-phosphoglycerate kinase from Methanothermus fervidus

  • Anselm Lehmacher
  • Reinhard Hensel
Original Articles

Abstract

The gene encoding 2-phosphoglycerate kinase (2PGK), which catalyses the first step in the biosynthesis of cyclic 2,3-diphosphoglycerate in methanogens, was cloned and sequenced from the hyperthermophilic Methanothermus fervidus. The 2pgk gene codes for 304 amino acids, corresponding to a relative molecular mass of 35040. The 2pgk mRNA was estimated to be 1600 nucleotides in size. Putative transcription signals and the ribosome-binding site of 2pgk are discussed. Production of 2PGK from M. fervidus in Es-cherichia coli reveals the same apparent molecular weights for the native enzyme and its denatured subunit as those shown by the 2PGK purified from M. fervidus. Also the kinetic parameters of 2PKG produced in E. coli correspond well with those from the enzyme isolated from the natural host M. fervidus.

Key words

Cyclic 2,3-diphosphoglycerate 2-phosphoglycerate kinase Methanogen 

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References

  1. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299Google Scholar
  2. Eckerskorn C, Mewes W, Goretzki H, Lottspeich F (1988) A new siliconized glass fiber as support for protein chemical analysis of electroblotted proteins. Eur J Biochem 176:509–519Google Scholar
  3. Evans JNS, Tolman CT, Kanodia S, Roberts MF (1985) 2,3-Cyclopyrophosphate in methanogens: Evidence by 13C NMR spectroscopy for a role in carbohydrate metabolism. Biochemistry 24:5693–5698Google Scholar
  4. Fabry S, Hensel R (1988) Primary structure of glyceraldehyde-3-phosphate dehydrogenase deduced from the nucleotide sequence of the thermophilic archaebacterium Methanothermus fervidus. Gene 64:189–197Google Scholar
  5. Fabry S, Lehmacher A, Bode W, Hensel R (1988) Expression of the glyceraldehyde-3-phosphate dehydrogenase gene from the extremely thermophilic archaebacterium Methanothermus fervidus in E. coli. FEBS Lett 237:213–217Google Scholar
  6. Fabry S, Heppner P, Dietmaier W, Hensel R (1990) Cloning and sequencing the gene encoding 3-phosphoglycerate kinase from mesophilic Methanobacterium bryantii and thermophilic Methanothermusfervidus. Gene 91:19–25Google Scholar
  7. Gorris LGM, Korteland J, Derksen RJAM, van der Drift C, Vogels GD (1990) Quantification of cyclic 2,3-diphosphoglycerate from methanogenic bacteria by isotachophoresis. J Chromatogr 504:421–428Google Scholar
  8. Haas ES, Brown JW, Daniels CJ, Reeve JN (1990) Genes encoding the 7S RNA and tRNASer, are linked to one of the two rRNA operons in the genome of the extremely thermophilic archaebacterium Methanothermus fervidus. Gene 90:51–59Google Scholar
  9. Hausner W, Frey G, Thomm M (1991) Control regions of an archaeal gene, a TATA box and an initiator element promote cell-free transcription of the tRNAVal gene of Methanococcus vannield. Mol Biol 222:495–508Google Scholar
  10. Hensel R, König H (1988) Thermoadaptation of methanogenic bacteria by intracellular ion concentration. FEMS Microbiol Lett 49: 75–79Google Scholar
  11. Huber R, Kurr M, Jannasch HW, Stetter KO (1989) A novel group of abyssal methanogenic archaebacteria (Methanopyrus) growing at 110° C. Nature 342: 833–834Google Scholar
  12. Kabsch W, Sander C (1983) Dictionary of protein secondary structure: Pattern recognition of hydrogen-bond and geometrical features. Biopolymers 22:2577–2637Google Scholar
  13. Kanodia S, Roberts MF (1983) Methanophosphagen: Unique cyclic pyrophosphate isolated from Methanobacterium thermoautotrophicum. Proc Natl Acad Sci USA 80:5217–5221Google Scholar
  14. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227:680–685Google Scholar
  15. Lehmacher A, Hensel R (1990) Cyclic 2,3-diphosphoglycerate, protein stabilizer from thermophilic archaebacteria: synthesis and function. (DECHEMA Biotechnology Conferences 4) VCH Verlagsgesellschaft, Weinheim, pp 415–418Google Scholar
  16. Lehmacher A, Vogt AB, Hensel R (1990) Biosynthesis of cyclic 2,3-diphosphoglycerate: Isolation and characterization of 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus. FEBS Lett 272:94–98Google Scholar
  17. Martin RG, Ames BN (1961) A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. Biol Chem 236:1372–1379Google Scholar
  18. Reiter WD, Palm P, Zillig W (1988) Transcription termination in the archaebacterium Sulfolobus: signal structures and linkage to transcription initiation. Nucleic Acids Res 16:2445–2459Google Scholar
  19. 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 154:584–588Google Scholar
  20. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  21. Saraste M, Sibbald PR, Wittinghofer A (1990) The P-loop — a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci 15:430–434Google Scholar
  22. Seely RJ, Fahrney DE (1983) A novel diphospho-P,P′-diester from Methan obacterium thermoautotrophicum. J Biol Chem 258:10835–10838Google Scholar
  23. Selden RF (1989) Analysis of RNA by Northern hybridization. In: Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith JA, Struhl K (eds) Current protocols in molecular biology. John Wiley and Sons, New York, pp 4.9.1–4.9.8Google Scholar
  24. Short JM, Fernandez JM, Sorge JA, Huse WD (1988) 1ZAP: A bacteriophage λ expression vector with in vivo excision properties. Nucleic Acids Res 16:7583–7600Google Scholar
  25. Stetter KO, Thomm M, Winter J, Wildgruber G, Huber H, Zillig W, Janécovic D, König H, Palm P, Wunderl S (1981) Methanothermus fervidus, sp. nov., a novel extremely thermophilic methanogen isolated from icelandic hot spring. Zentralbl Bakteriol Hyg [C] 2:166–178Google Scholar
  26. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89Google Scholar
  27. Tolman C, Kanodia S, Roberts MF, Daniels L (1986) 31P-NMR spectra of methanogens: 2,3-cyclopyrophosphoglycerate is detectable only in methanogenic strains. Biochim Biophys Acta 886:345–352Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Anselm Lehmacher
    • 1
  • Reinhard Hensel
    • 1
  1. 1.Max-Planck-Institut für Biochemie, Am KlopferspitzMartinsriedGermany

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