Skip to main content
SpringerLink
Log in

Effect of Structure Variations in the Inter-subunit Contact Zone on the Activity and Allosteric Regulation of Inorganic Pyrophosphatase from Mycobacterium tuberculosis

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

Hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) has a number of structural and functional features that distinguish it from homologous enzymes widely occurring in living organisms. In particular, it has unusual zones of inter-subunit contacts and lacks the N-terminal region common for other PPases. In this work, we constructed two mutant forms of the enzyme, Ec-Mt-PPase and R14Q-Mt-PPase. In Ec-Mt-PPase, the missing part of the polypeptide chain was compensated with a fragment of PPase from Escherichia coli (Ec-PPase). In R14Q-Mt-PPase, a point mutation was introduced to the contact interface between the two trimers of the hexamer. Both modifications significantly improved the catalytic activity of the enzyme and abolished its inhibition by the cofactor (Mg2+ ion) excess. Activation of Mt-PPase by low (∼10 µM) concentrations of ATP, fructose-1-phosphate, L-malate, and non-hydrolyzable substrate analogue methylene bisphosphonate (PCP) was observed. At concentrations of 100 µM and higher, the first three compounds acted as inhibitors. The activating effect of PCP was absent in both mutant forms, and the inhibitory effect of fructose-1-phosphate was absent in Ec-Mt-PPase. The effects of other modulators varied only quantitatively among the mutants. The obtained data indicate the presence of allosteric sites in Mt-PPase, which are located in the zones of inter-subunit contact or associated with them.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

PPase:

inorganic pyrophosphatase

Ec-PPase:

PPase from Escherichia coli

Mt-PPase:

inorganic pyrophosphatase from Mycobacterium tuberculosis

Ec-Mt-PPase:

chimeric enzyme in which Mt-PPase polypeptide chain is extended at the N-terminus with residues 1–12 of Ec-PPase

R14Q-Mt-PPase:

mutant variant of Mt-PPase with Arg14 replaced by Gln

PCP:

methylene bisphosphonate

References

  1. Baykov, A. A., Cooperman, B. S., Goldman, A., and Lahti, R. (1999) Cytoplasmic inorganic pyrophosphatase, in Inorganic Polyphosphates, Springer, pp. 127–150.

  2. Cooperman, B. S., Baykov, A. A., and Lahti, R. (1992) Evolutionary conservation of the active site of soluble inorganic pyrophosphatase, Trends Biochem. Sci., 17, 262–266.

    Article  CAS  Google Scholar 

  3. Samygina, V. R. (2016) Inorganic pyrophosphatases: structural diversity serving the function, Russ. Chem. Rev., 85, 464–476.

    Article  CAS  Google Scholar 

  4. Heikinheimo, P., Tuominen, V., Ahonen, A.-K., Teplyakov, A., Cooperman, B., Baykov, A., Lahti, R., and Goldman, A. (2001) Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase, Proc. Natl. Acad. Sci. USA, 98, 3121–3126.

    Article  CAS  Google Scholar 

  5. Vijayan, M. (2005) Structural biology of mycobacterial proteins: the Bangalore effort, Tuberculosis (Edinb), 85, 357–366.

    Article  CAS  Google Scholar 

  6. Rodina, E. V., Valueva, A. V., Yakovlev, R. Y., Vorobyeva, N. N., Kulakova, I. I., Lisichkin, G. V., and Leonidov, N. B. (2015) Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity, Biointerphases, 10, 041005.

    Article  Google Scholar 

  7. Tammenkoski, M., Benini, S., Magretova, N. N., Baykov, A. A., and Lahti, R. (2005) An unusual, His-dependent family I pyrophosphatase from Mycobacterium tuberculosis, J. Biol. Chem., 280, 41819–41826.

    Article  CAS  Google Scholar 

  8. Pratt, A. C., Dewage, S. W., Pang, A. H., Biswas, T, Barnard-Britson, S., Cisneros, G. A., and Tsodikov, O. V. (2015) Structural and computational dissection of the catalytic mechanism of the inorganic pyrophosphatase from Mycobacterium tuberculosis, J. Struct. Biol., 192, 76–87.

    Article  CAS  Google Scholar 

  9. Harutyunyan, E. H., Oganessyan, V. Y., Oganessyan, N. N., Avaeva, S. M., Nazarova, T. I., Vorobyeva, N. N., Kurilova, S. A., Huber, R., and Mather, T. (1997) Crystal structure of holo inorganic pyrophosphatase from Escherichia coli at 1.9 A resolution. Mechanism of hydrolysis, Biochemistry, 36, 7754–7760.

    Article  CAS  Google Scholar 

  10. Pettersen, E. F., Goddard, T D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T E. (2004) UCSF Chimera — a visualization system for exploratory research and analysis, J. Comput. Chem., 25, 1605–1612.

    Article  CAS  Google Scholar 

  11. Rodina, E. V., Vainonen, L. P., Vorobyeva, N. N., Kurilova, S. A., Sitnik, T. S., and Nazarova, T. I. (2008) Metal cofactors play a dual role in Mycobacterium tuberculosis inorganic pyrophosphatase, Biochemistry (Moscow), 73, 897–905.

    Article  CAS  Google Scholar 

  12. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  13. Rowlands, M. G., Newbatt, Y. M., Prodromou, C., Pearl, L. H., Workman, P., and Aherne, W. (2004) High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity, Anal. Biochem., 327, 176–183.

    Article  CAS  Google Scholar 

  14. Schuck, P., Gillis, R. B., Besong, T. M., Almutairi, F., Adams, G. G., Rowe, A. J., and Harding, S. E. (2014) SEDFIT—MSTAR: molecular weight and molecular weight distribution analysis of polymers by sedimentation equilibrium in the ultracentrifuge, Analyst, 139, 79–92.

    Article  CAS  Google Scholar 

  15. Rodina, E. V., Vorobyeva, N. N., Kurilova, S. A., Sitnik, T. S., and Nazarova, T. I. (2007) ATP as effector of inorganic pyrophosphatase of Escherichia coli. The role of residue Lys112 in binding effectors, Biochemistry (Moscow), 72, 100–108.

    Article  CAS  Google Scholar 

  16. Vorobyeva, N. N., Kurilova, S. A., Anashkin, V. A., and Rodina, E. V. (2017) Inhibition of Escherichia coli inorganic pyrophosphatase by fructose-1-phosphate, Biochemistry (Moscow), 82, 953–956.

    Article  CAS  Google Scholar 

  17. Vainonen, Yu. P., Kurilova, S. A., and Avaeva, S. M. (2002) Hexameric, trimeric, dimeric, and monomeric forms of inorganic pyrophosphatase from Escherichia coli, Russ. J. Bioorg. Chem., 28, 385–391.

    Article  CAS  Google Scholar 

  18. Avaeva, S. M., Rodina, E. V., Kurilova, S. A., Nazarova, T. I., Vorobyeva, N. N., Harutyunyan, E. H., and Oganessyan, V. Y. (1995) Mg2+ activation of Escherichia coli inorganic pyrophosphatase, FEBS Lett., 377, 44–46.

    Article  CAS  Google Scholar 

  19. Sitnik, T., Vainonen, J., Rodina, E., Nazarova, T., Kurilova, S., Vorobyeva, N., and Avaeva, S. (2003) Effectory site in Escherichia coli inorganic pyrophosphatase is revealed upon mutation at the intertrimeric interface, IUBMB Life, 55, 37–41.

    Article  CAS  Google Scholar 

  20. Samygina, V. R., Moiseev, V. M., Rodina, E. V., Vorobyeva, N. N., Popov, A. N., Kurilova, S. A., Nazarova, T. I., Avaeva, S. M., and Bartunik, H. D. (2007) Reversible inhibition of Escherichia coli inorganic pyrophosphatase by fluoride: trapped catalytic intermediates in cryo-crystallographic studies, J. Mol. Biol., 366, 1305–1317.

    Article  CAS  Google Scholar 

  21. Avaeva, S. M., Vorobyeva, N. N., Kurilova, S. A., Nazarova, T. I., Polyakov, K. M., Rodina, E. V., and Samygina, V. R. (2000) Mechanism of Ca2+-induced inhibition of Escherichia coli inorganic pyrophosphatase, Biochemistry (Moscow), 65, 373–387.

    CAS  Google Scholar 

  22. Vainonen, J. P., Vorobyeva, N. N., Rodina, E. V., Nazarova, T. I., Kurilova, S. A., Skoblov, J. S., and Avaeva, S. M. (2005) Metal-free PPi activates hydrolysis of MgPPi by an Escherichia coli inorganic pyrophosphatase, Biochemistry (Moscow), 70, 69–78.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors are grateful to P. V. Kalmykov and N. N. Magretova for performing analytical ultracentrifugation, N. N. Vorobyeva for providing EcPPase, and M. V. Serebryakov for performing protein mass spectrometry analysis. MALDI-mass spectrometer was used in the framework of the Program of Moscow State University Development.

Author information

Authors and Affiliations

  1. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991, Moscow, Russia

    R. S. Romanov, S. A. Kurilova & A. A. Baykov

  2. Faculty of Chemistry, Lomonosov Moscow State University, 119991, Moscow, Russia

    E. V. Rodina

Authors
  1. R. S. Romanov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. A. Kurilova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. A. Baykov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E. V. Rodina
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to E. V. Rodina.

Additional information

Russian Text © The Author(s), 2020, published in Biokhimiya, 2020, Vol. 85, No. 3, pp. 378–386.

Conflict of interest

The authors declare no conflict of interest in financial or any other sphere.

Ethical approval

This article does not contain description of studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Romanov, R.S., Kurilova, S.A., Baykov, A.A. et al. Effect of Structure Variations in the Inter-subunit Contact Zone on the Activity and Allosteric Regulation of Inorganic Pyrophosphatase from Mycobacterium tuberculosis. Biochemistry Moscow 85, 326–333 (2020). https://doi.org/10.1134/S0006297920030086

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297920030086

Keywords

Search

Navigation