Archives of Microbiology

, Volume 179, Issue 5, pp 368–376 | Cite as

Rhodobacter sphaeroides has a family II pyrophosphatase: comparison with other species of photosynthetic bacteria

  • Heliodoro Celis
  • Bernardo Franco
  • Silvia Escobedo
  • Irma Romero
Original Paper


The cytoplasmic pyrophosphatase from Rhodobacter sphaeroides was purified and characterized. The enzyme is a homodimer of 64 kDa. The N-terminus was sequenced and used to obtain the complete pyrophosphatase sequence from the preliminary genome sequence of Rba. sphaeroides, showing extensive sequence similarity to family II or class C pyrophosphatases. The enzyme hydrolyzes only Mg-PPi and Mn-PPi with a K m of 0.35 mM for both substrates. It is not activated by free Mg 2+, in contrast to the cytoplasmic pyrophosphatase from Rhodospirillum rubrum, and it is not inhibited by NaF, methylendiphosphate, or imidodiphosphate. This work shows that Rba. sphaeroides and Rhodobacter capsulatus cytoplasmic pyrophosphatases belong to family II, in contrast to Rsp. rubrum, Rhodopseudomonas palustris, Rhodopseudomonas gelatinosa, and Rhodomicrobium vannielii cytoplasmic pyrophosphatases which should be classified as members of family I. This is the first report of family II cytoplasmic pyrophosphatases in photosynthetic bacteria and in a gram-negative organism.


Photosynthetic bacteria Rhodobacter sphaeroides Rhodospirillum rubrum Rhodobacter capsulatus Family II inorganic pyrophosphatase 







3-[Cyclohexylamino]-1-propanesulfonic acid



This work was supported in part by grant IN 216401 from DGAPA. We thank the Molecular Biology Unit of the IFC for sequencing genes, and primer synthesis. The authors would like to thank Dr. Mark West for his assistance with protein purification and suggestions regarding the manuscript. We also thank Dr. Georges Dreyfus for critically reading the manuscript.


  1. Ahn S, Milner AJ, Fütterer K, Konopka M, Ilias M, Young TW, White S (2001) The "open" and "closed" structures of the type-C inorganic pyrophosphatase from Bacillus subtilis and Streptococcus gordonii. J Mol Biol 313:797–811CrossRefPubMedGoogle Scholar
  2. Avaeva SM, Rodina EU, Kurilova SA, Nazarova TI, Vorovyeva NN (1996) Effect of D42 N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity of Mg2+ binding. FEBS Lett 392:91–94CrossRefPubMedGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  4. Celis H, Romero I (1987) The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase of Rhodospirillum rubrum: effects of pH and divalent cations. J Bioenerg Biomembr 19:255–272PubMedGoogle Scholar
  5. Chen J, Brevet A. Fromant M, Leveque F, Schmitter J M, Blanquet S, Plateu P. (1990) Pyrophosphatase is essential for growth in Escherichia coli. J Bacteriol 172:5686–5689PubMedGoogle Scholar
  6. Cohen-Bazire G, Sistrom W R, Stainer RY (1957) The kinetics studies of pigment synthesis by non-sulfur purple bacteria. J Cell Comp Physiol.49:25–68Google Scholar
  7. Cooperman BS, Baykov A-A, Lathi R (1992) Evolutionary conservation of the active site of soluble inorganic pyrophosphatase. Trends Biochem Sci 17:262–266PubMedGoogle Scholar
  8. Fabrichniy IP, Kasho VN, Hyytiä T, Salminen T, Halonen P, Dudarenkov VYu, Heikinheimo P, Chernyak UYa, Goldman A, Lathi R, Cooperman BS, Baykov AA (1997) Structural and functional consequences of substitution at the tyrosine 55-lysine 104 hydrogen bond in Escherichia coli inorganic pyrophosphatase. Biochemistry 36:7746–7753CrossRefPubMedGoogle Scholar
  9. Gupta RS (1998) Protein phylogenies and signature sequences: A reappraisal of evolutionary relationships among archaebacteria, eubacteria, and eukaryotes. Microbial Mol Biol Rev 62:1435–1491Google Scholar
  10. Harutyunyan EH, Kuranova IP, Vainshtein BK, Höhne WE, Lamzin VS, Dauter Z, Teplyakov AV, Wilson KS (1996) X-ray structure of yeast inorganic pyrophosphatase complexed with manganese and phosphate. Eur J Biochem 239:220–228PubMedGoogle Scholar
  11. Harutyunyan EH, Oganessyan VYu, Oganessyan NN, Avaeva SM, Nazarova TI, Vorobyeva NN, Kurilova SA, Huber R, Mather T (1997) Crystal structure of holo inorganic pyrophosphatase from Escherichia coli at 1.9 Å resolution mechanism of hydrolysis. Biochemistry 36:7754–7760CrossRefPubMedGoogle Scholar
  12. Heikinheimo P, Lehtonen J, Baykov A, Lahti R, Cooperman BS, Goldman A (1996) The structural basis for pyrophosphatase catalysis. Structure 4:1491–1508PubMedGoogle Scholar
  13. Kankare J, Salminen T, Lahti R, Cooperman BS, Baykov AA, Goldman A (1996) Structure of Escherichia coli inorganic pyrophosphatase at 2.2 Å resolution. Acta Crystallogr D52:551–563Google Scholar
  14. Käpylä J, Hyytiä T, Lahti R, Goldman A, Baykov AA, Cooperman BS (1995) Effect of D97E substitution on the kinetic and thermodynamic properties of Escherichia coli inorganic pyrophosphatase. Biochemistry 34:792–800PubMedGoogle Scholar
  15. Klemme JH, Gest H (1971) Regulation of the cytoplasmic pyrophosphatase of Rhodospirillum rubrum. Eur J Biochem 22:529–537PubMedGoogle Scholar
  16. Klemme JH, Klemme B, Gest H (1971) Catalytic properties and regulatory diversity of inorganic pyrophosphatases from photosynthetic bacteria. J Bacteriol 108:1122–1128PubMedGoogle Scholar
  17. Konopka MA, White SA,Young TW (2002) Bacillus subtilis inorganic pyrophosphatase: The C-terminal signature sequence is essential for enzyme activity and conformational integrity. Biochem Biophys Res Commun 290:806–812CrossRefPubMedGoogle Scholar
  18. Kornberg A (1962) On the metabolic significance of phosphorolytic and pyrophosphorolytic reactions. In: Kasha H, Pullman B (eds) Horizons in biochemistry. Academic, New York, pp 251–264Google Scholar
  19. Kuhn NJ, Ward S (1998) Purification, properties and multiple forms of a manganese-activated inorganic pyrophosphatase from Bacillus subtilis. Arch Biochem Biophys 354:47–56CrossRefPubMedGoogle Scholar
  20. Kuhn NJ, Wadeson A, Ward S, Young TW (2000) Methanococcus jannaschii ORF mj0608 codes for a class C inorganic pyrophosphatase protected by Co2+ or Mn2+ ions against fluoride inhibition. Arch Biochem Biophys 379:292–298CrossRefPubMedGoogle Scholar
  21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  22. Lundblad RL (1991) Chemical reagents for protein modification, 2nd edn. CRC, Boca Raton, FloridaGoogle Scholar
  23. Lundin M, Baltscheffsky H, Ronne H (1991) Yeast-ppa2 gene encodes a mitochondrial inorganic pyrophosphatase that is essential for mitochondrial function. J Biol Chem 266:12168–12172PubMedGoogle Scholar
  24. Martell A, Sillén LG (1971) Stability constants of metal-ion complexes: supplement no. 1, special publication no.25. The Chemical Society, LondonGoogle Scholar
  25. Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038PubMedGoogle Scholar
  26. Merckel MC, Fabrichniy IP, Salminen A, Kalkkinen N, Baykov AA, Lahti R, Goldman A (2001) Crystal structure of Streptococcus mutans pyrophosphatase: a new fold for an old mechanism. Structure 9:289–297CrossRefPubMedGoogle Scholar
  27. Morrisey J H (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117:307–310PubMedGoogle Scholar
  28. Ordaz H, Sosa A, Romero I, Celis H (1992) Thermostability and activation by divalent cations of the membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum. Int J Biochem 24:1633–1638CrossRefGoogle Scholar
  29. Parfenyev AN, Salminen A, Halonen P, Hachimori A, Baykov AA, Lahti R (2001) Quaternary structure and metal ion requirement of family II pyrophosphatases from Bacillus subtilis, Streptococcus gordonii and Streptococcus mutans. J. Biol. Chem. 276:24511–24518Google Scholar
  30. Pohjanjoki P, Lahti R, Goldman A, Cooperman BS (1998) Evolutionary conservation of enzymatic catalysis: quantitative comparison of the effects of mutation of aligned residues in Saccharomyces cereviseae and Escherichia coli inorganic pyrophosphatases on enzymatic activity. Biochemistry 37:1754–1761CrossRefPubMedGoogle Scholar
  31. Romero I, Gómez-Priego A, Celis H (1991) A membrane-bound pyrophosphatase from respiratory membranes of Rhodospirillum rubrum. J Gen Microbiol 137:2611–2616Google Scholar
  32. Romero I, García-Contreras R, Celis H (2003) Rhodospirillum rubrum has a family I pyrophosphatase: purification, cloning, and sequencing. Arch Microbiol DOI 10.1007/s00203-003-0537-4Google Scholar
  33. Salminen T, Käpylä J, Heikinheimo P, Kankare J, Goldman A, Heinonen J, Baykov AA, Cooperman BS, Lahti R (1995) Structure and function analysis of Escherichia coli inorganic pyrophosphatase is a hydroxide ion the key to catalysis? Biochemistry 34:782–791PubMedGoogle Scholar
  34. Shintani T, Uchiumi T, Yonezawa T, Salminen A, Baykov A, Lahti R, Hachimori A (1998) Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis: evidence for a new family of enzymes. FEBS Lett 439:263–266CrossRefPubMedGoogle Scholar
  35. Shizawa N, Uchiumi T, Taguchi J, Kisseleva NA, Baykov AA, Lahti R, Hachimori A (2001) Directed mutagenesis studies of the C-terminal fingerprint region of Bacillus subtilis pyrophosphatase. Eur J Biochem 268: 5771–5775CrossRefPubMedGoogle Scholar
  36. Sivula T, Salminen A, Parfenyev AN, Pohjanjoki P, Goldman A, Cooperman BS, Baykov A. Lahti R (1999) Evolutionary aspects of inorganic pyrophosphatase. FEBS Lett 454:75–80CrossRefPubMedGoogle Scholar
  37. Sonnewald U (1992) Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning. Plant J 2:571–581CrossRefPubMedGoogle Scholar
  38. Sumner JB (1944) A method for the colorimetric determination of phosphorous. Science 100:413–415Google Scholar
  39. Volk SE, Dudarenkou VY, Käpylä J, Kasho VN, Voloshima OA, Salminen T, Goldman A, Lahti A, Baykov AA, Cooperman BS (1996) Effect of E20D substitution in the active site of Escherichia coli inorganic pyrophosphatase on its quaternary and catalytic properties. Biochemistry 35:4662–4669CrossRefPubMedGoogle Scholar
  40. Young T W, Kuhn NJ, Wadeson A, Ward S, Burges D, Cooke GD (1998) Open reading frame yybQ of Bacillus subtilis encodes a soluble inorganic pyrophosphatase with distinctive properties: the first of a new class of pyrophosphatases? Microbiology 144:2563–2571PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Heliodoro Celis
    • 1
  • Bernardo Franco
    • 1
  • Silvia Escobedo
    • 1
  • Irma Romero
    • 2
  1. 1.Instituto de Fisiología CelularUniversidad Nacional Autónoma de MéxicoMéxico
  2. 2.Departamento de Bioquímica, Facultad de MedicinaUniversidad Nacional Autónoma de MéxicoMéxico

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