Biochemistry (Moscow)

, Volume 77, Issue 3, pp 288–295 | Cite as

Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060

  • O. N. Rozova
  • V. N. KhmeleninaEmail author
  • Y. A. Trotsenko


The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His6-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.

Key words

pyrophosphate-dependent 6-phosphofructokinase methylotrophic bacteria sedoheptulose-1,7-bisphosphatase glycolysis gluconeogenesis 



ATP-dependent 6-phosphofructokinase






isopropyl β-D-thiogalactopyranoside


Luria-Bertani medium




pyrophosphate-dependent 6-phosphofructokinase






ribulose monophosphate






Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bapteste, E., Moreira, D., and Philippe, H. (2003) Gene, 318, 185–191.PubMedCrossRefGoogle Scholar
  2. 2.
    Siebers, B., Klenk, H. P., and Hensel, R. (1998) J. Bacteriol., 180, 2137–2143.PubMedGoogle Scholar
  3. 3.
    Chi, A., and Kemp, R. G. (2000) J. Biol. Chem., 275, 35677–35679.PubMedCrossRefGoogle Scholar
  4. 4.
    Trotsenko, Y. A., and Shishkina, V. N. (1990) FEMS Microbiol. Lett., 87, 267–271.CrossRefGoogle Scholar
  5. 5.
    Alves, A. M. C. R., Euverink, G. J. W., Hektor, H. J., Hessels, G. I., van der Vlag, J., Vrijbloed, J. W., Hondmann, D., Visser, J., and Dijkhuizen, L. (1994) J. Bacteriol., 176, 6827–6835.PubMedGoogle Scholar
  6. 6.
    Beschastny, A. P., Khmelenina, V. N., Rozova, O. N., and Trotsenko, Y. A. (2008) Mikrobiologiya, 77, 591–594.Google Scholar
  7. 7.
    Taylor, S. C., Dalton, H., and Dow, C. S. (1981) J. Gen. Microbiol., 122, 89–94.Google Scholar
  8. 8.
    Eshinimaev, B. T., Medvedkova, K. A., Khmelenina, V. N., Suzina, N. E., Osipov, G. A., Lysenko, A. M., and Trotsenko, Y. A. (2004) Mikrobiologiya, 73, 530–539.Google Scholar
  9. 9.
    Trotsenko, Y. A., and Murrell, J. C. (2008) Adv. Appl. Microbiol., 63, 183–229.PubMedCrossRefGoogle Scholar
  10. 10.
    Beschastny, A. P., Sokolov, A. P., Khmelenina, V. N., and Trotsenko, Y. A. (1992) Biokhimiya, 57, 1215–1221.Google Scholar
  11. 11.
    Reshetnikov, A. S., Rozova, O. N., Khmelenina, V. N., Mustakhimov, I. I., Beschastny, A. P., Murrell, J., and Trotsenko, Y. A. (2008) FEMS Microbiol. Lett., 288, 202–210.PubMedCrossRefGoogle Scholar
  12. 12.
    Rozova, O. N., Khmelenina, V. N., Vuilleumier, S., and Trotsenko, Y. (2010) Res. Microbiol., 161, 861–868.PubMedCrossRefGoogle Scholar
  13. 13.
    Khmelenina, V. N., Rozova, O. N., and Trotsenko, Y. A. (2011) Meth. Enzymol., 495, 1–14.PubMedGoogle Scholar
  14. 14.
    Khmelenina, V. N., Beschastny, A. P., Gayazov, R. R., and Trotsenko, Y. A. (1994) Mikrobiologiya, 63, 95–98.Google Scholar
  15. 15.
    Sy, A., Giraud, E., Jourand, P., Garcia, N., Willems, A., de Lajudie, P., Prin, Y., Neyra, M., Gillis, M., Boivin-Masson, C., and Dreyfus, B. (2001) J. Bacteriol., 183, 214–220.PubMedCrossRefGoogle Scholar
  16. 16.
    Kalyuzhnaya, M., Khmelenina, V. N., Kotelnikova, S., Holmquist, L., Pedersen, K., and Trotsenko, Y. A. (1999) Syst. Appl. Microbiol., 22, 565–572.PubMedCrossRefGoogle Scholar
  17. 17.
    Baker, R. T., Catanzariti, A.-M., Karunasekara, Y., Soboleva, T. A., Sharwood, R., Whitney, S., and Board, P. (2005) Meth. Enzymol., 398, 540–554.PubMedCrossRefGoogle Scholar
  18. 18.
    Laemmli, U. K. (1970) Nature, 227, 680–685.PubMedCrossRefGoogle Scholar
  19. 19.
    Slater, G. G. (1969) Anal. Chem., 41, 1039–1041.PubMedCrossRefGoogle Scholar
  20. 20.
    Shacterle, G. R., and Pollack, R. L. (1973) Anal. Biochem., 51, 654–657.CrossRefGoogle Scholar
  21. 21.
    Thomson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997) Nucleic Acids Res., 24, 4876–4882.CrossRefGoogle Scholar
  22. 22.
    Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) Molecular Biology and Evolution, 10.1093/molbev/ msm092.Google Scholar
  23. 23.
    Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: a Laboratory Manual, 3rd Edn., Cold Spring Harbor Laboratory, N.-Y.Google Scholar
  24. 24.
    Pfleiderer, C., and Klemme, J. H. (1980) Z. Naturforsch., 35, 229–238.Google Scholar
  25. 25.
    Bertagnolli, B. L., Younathan, E. S., Voll, R. J., Pittman, C. E., and Cook, P. F. (1986) Biochemistry, 25, 4674–4681.PubMedCrossRefGoogle Scholar
  26. 26.
    Mertens, E., van Schaftingen, E., and Muller, M. (1989) Mol. Biochem. Parasitol., 37, 183–190.PubMedCrossRefGoogle Scholar
  27. 27.
    Jourand, P., Giraud, E., Bena, G., Sy, A., Willems, A., Gillis, M., Dreyfus, B., and de Lajudie, P. (2004) Int. J. System. Evol. Microbiol., 54, 2269–2273.CrossRefGoogle Scholar
  28. 28.
    Susskind, B. M., Warren, L. G., and Reeves, R. E. (1982) Biochem. J., 204, 191–196.PubMedGoogle Scholar
  29. 29.
    Loftus, B., Anderson, I., Davies, R., Alsmark, U. C. M., Samuelson, J., Amedeo, P., Roncaglia, P., Berriman, M., Hirt, R. P., Mann, B. J., Nozaki, T., Suh, B., Pop, M., Duchene, M., Ackers, J., Tannich, E., Leippe, M., Hofer, M., Bruchhaus, I., Willhoeft, U., Bhattacharya, A., Chillingworth, T., Churcher, C., Hance, Z., Harris, B., Harris, D., Jagels, K., Moule, S., Mungall, K., Ormond, D., Squares, R., Whitehead, S., Quail, M. A., Rabbinowitsch, E., Norbertczak, H., Price, C., Wang, Z., Guillen, N., Gilchrist, C., Stroup, S. E., Bhattacharya, S., Lohia, A., Foster, P. G., Sicheritz-Ponten, T., Weber, C., Singh, U., Mukherjee, C., El-Sayed, N. M., Petri, W. A., Jr., Clark, C. G., Embley, T. M., Barrell, B., Fraser, C. M., and Hall, N. (2005) Nature, 433, 865–868.PubMedCrossRefGoogle Scholar
  30. 30.
    Muller, M., Lee, J. A., Gordon, P., Gaasterland, T., and Sensen, C. W. (2001) J. Bacteriol., 183, 6714–6716.PubMedCrossRefGoogle Scholar
  31. 31.
    Roslev, P., and King, G. M. (1995) Appl. Environ. Microbiol., 61, 1563–1570.PubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2012

Authors and Affiliations

  • O. N. Rozova
    • 1
  • V. N. Khmelenina
    • 1
    Email author
  • Y. A. Trotsenko
    • 1
  1. 1.Skryabin Institute of Biochemistry and Physiology of MicroorganismsRussian Academy of SciencesPushchino, Moscow RegionRussia

Personalised recommendations