Skip to main content

Structural Organization of the Active Center of Unmodified Recombinant Sulfatase from the Mycelial Fungi Fusarium proliferatum LE1

Abstract

Sulfatases catalyze the hydrolysis of sulfuric acid esters and play a key role in a number of biological processes of both higher eukaryotes and prokaryotes. According to literature data, for the implementation of catalysis, some representatives of this group of enzymes require posttranslational modification of serine or cysteine residues in the active center into the unique amino acid Cα-formylglycine. Nevertheless, it is confirmed that even in the absence of this modification, some sulfatases are capable of catalyzing the hydrolysis of sulfoesters. In this work, we studied the structural and functional features of active recombinant sulfatase from the mycelial fungus Fusarium proliferatum LE1, which contains a cysteine residue in the active center. A theoretical atomic model of the enzyme was first constructed, the structural organization of its active center was determined, and key amino acid residues involved in the binding of p-nitrophenyl sulfate and in the reaction of its hydrolysis were identified. Point amino acid substitutions of the identified residues led to inactivation of the enzyme. In particular, mutant forms of the enzyme with the replacement of catalytic cysteine by serine and threonine containing a hydroxyl group in the side chain completely lost activity, which indicates the direct participation of the mercapto group of cysteine in the hydrolysis reaction.

This is a preview of subscription content, access via your institution.

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.

REFERENCES

  1. Stressler, T., Seitl, I., Kuhn, A., and Fischer, L., Appl. Microbiol. Biotechnol., 2016, vol. 100, no. 21, pp. 9053–9067.

    CAS  PubMed  Google Scholar 

  2. Hanson, S.R., Best, M.D., and Wong, C.-H., Angew. Chem., Int. Ed. Engl., 2004, vol. 43, no. 43, pp. 5736–5763.

    CAS  Google Scholar 

  3. Diez-Roux, G. and Ballabio, A., Annu. Rev. Genomics Hum. Genet., 2005, vol. 6, no. 1, pp. 355–379.

    CAS  PubMed  Google Scholar 

  4. Rižner, T.L., Front. Pharmacol., 2016, vol. 7, p. 30.

    PubMed  PubMed Central  Google Scholar 

  5. Shvetsova, S.V. and Kul’minskaya, A.A., Vestn. Mosk. Univ., 2018, vol. 59, no. 4, pp. 243–256.

    CAS  Google Scholar 

  6. Toesch, M., Schober, M., and Faber, K., Appl. Microbiol. Biotechnol., 2014, vol. 98, no. 4, pp. 1485–1496.

    CAS  PubMed  Google Scholar 

  7. Hossain, Md.M., Kawarabayasi, Y., Kimura, M., and Kakuta, Y., J. Biochem. (Tokyo), 2009, vol. 146, no. 6, pp. 767–769.

    CAS  Google Scholar 

  8. Markham, P., Robson, G.D., Bainbridge, B.W., and Trinci, A.P., FEMS Microbiol. Rev., 1993, vol. 10, nos. 3–4, pp. 287–300.

    CAS  PubMed  Google Scholar 

  9. Barbeyron, T., Brillet-Gueguen, L., Carre, W., Carriere, C., Caron, C., Czjzek, M., Hoebeke, M., and Michel, G., PLoS One, 2016, vol. 11, no. 10, e0164 846.

    Google Scholar 

  10. http://abims.sb-roscoff.fr/sulfatlas/index.html.

  11. Miech, C., Dierks, T., Selmer, T., von Figura, K., and Schmidt, B., J. Biol. Chem., 1998, vol. 273, no. 9, pp. 4835–4837.

    CAS  PubMed  Google Scholar 

  12. Schmidt, B., Selmer, T., Ingendoh, A., and von Figura, K., Cell, 1995, vol. 82, no. 2, pp. 271–278.

    CAS  PubMed  Google Scholar 

  13. Olguin, L.F., Askew, S.E., O’Donoghue, A.C., and Hollfelder, F., J. Am. Chem. Soc., 2008, vol. 130, no. 49, pp. 16 547–16 555.

    Google Scholar 

  14. Recksiek, M., Selmer, T., Dierks, T., Schmidt, B., and von Figura, K., J. Biol. Chem., 1998, vol. 273, no. 11, pp. 6096–6103.

    CAS  PubMed  Google Scholar 

  15. Stressler, T., Reichenberger, K., Glück, C., Leptihn, S., Pfannstiel, J., Swietalski, P., Kuhn, A., Seitl, I., and Fischer, L., Appl. Microbiol. Biotechnol., 2018, vol. 102, no. 6, pp. 2709–2721.

    CAS  PubMed  Google Scholar 

  16. Williams, S.J., Denehy, E., and Krenske, E.H., Org. Chem., 2014, vol. 79, no. 5, pp. 1995–2005.

    CAS  Google Scholar 

  17. Sánchez -Romero, J.J. and Olguin, L.F., Biochem. Biophys. Rep., 2015, vol. 3, pp. 161–168.

    PubMed  PubMed Central  Google Scholar 

  18. Korban, S.A., Bobrov, K.S., Maynskova, M.A., Naryzhny, S.N., Vlasova, O.L., Eneyskaya, E.V., and Kulminskaya, A.A., Protein Eng. Des. Sel., 2017, vol. 30, no. 7, pp. 477–488.

    CAS  PubMed  Google Scholar 

  19. van Loo, B., Schober, M., Valkov, E., Heberlein, M., Bornberg-Bauer, E., Faber, K., Hyvönen, M., and Hollfelder, F., J. Mol. Biol., 2018, vol. 430, no. 7, pp. 1004–1023.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Distler, H., Angew. Chem., Int. Ed. Engl., 1967, vol. 6, no. 6, pp. 544–553.

    CAS  Google Scholar 

  21. Dierks, T., Miech, C., Hummerjohann, J., Schmidt, B., Kertesz, M.A., and von Figura, K., J. Biol. Chem., 1998, vol. 273, no. 40, pp. 25 560–25 564.

    Google Scholar 

  22. Cartmell, A., Lowe, E.C., Basle, A., Firbank, S.J., Ndeh, D.A., Murray, H., Terrapon, N., Lombard, V., Henrissat, B., and Turnbull, J.E., Proc. Natl. Acad. Sci. U. S. A., 2017, vol. 114, no. 27, pp. 7037–7042.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Varfolomeev, S.D., Gariev, I.A., and Uporov, I.V., Usp. Khim., 2005, vol. 74, no. 1, pp. 67–83.

    Google Scholar 

  24. Nagahara, N., J. Amino Acids, 2010, vol. 2011, p. 709 404.

    Google Scholar 

  25. Huggins, C. and Smith, D.R., J. Biol. Chem., 1947, vol. 170, no. 1, pp. 391–398.

    CAS  Google Scholar 

  26. Lazur’evskii, G.V., Terent’eva, I.V., and Shamshurin, A.A., Prakticheskie raboty po khimii prirodnykh soedinenii (Practical Work in Chemistry of Natural Compounds), 2nd ed., Moscow: Vysshaya Shkola, 1966.

  27. Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Gallo Cassarino, T., Bertoni, M., and Bordoli, L., Nucleic Acids Res., 2014, vol. 42 (Web Server issue), pp. W252–W258.

  28. Abagyan, R., Totrov, M., and Kuznetsov, D., J. Comput. Chem., 1994, vol. 15, no. 5, pp. 488–506.

    CAS  Google Scholar 

  29. Halgren, T.A., J. Comput. Chem., 1996, vol. 17, nos. 5–6, pp. 490–519.

    CAS  Google Scholar 

  30. Fernandez-Recio, J., Totrov, M., and Abagyan, R., Proteins, 2003, vol. 52, no. 1, pp. 113–117.

    CAS  PubMed  Google Scholar 

  31. Totrov, M. and Abagyan, R., Proteins, 1997, suppl., pp. 215–220.

  32. Salomon-Ferrer, R., Case, D.A., and Walker, R.C., WIREs Comput.Mol. Sci., 2013, vol. 3, no. 2, pp. 198–210.

    CAS  Google Scholar 

  33. Li, P., Song, L.F., and Merz, K.M., J. Chem. Theory Comput., 2015, vol. 11, no. 4, pp. 1645–1657.

    CAS  PubMed  Google Scholar 

  34. Li, P., Song, L.F., and Merz, K.M., J. Phys. Chem. B, 2015, vol. 119, no. 3, pp. 883–895.

    CAS  PubMed  Google Scholar 

  35. http://nebasechanger.neb.com/.

Download references

ACKNOWLEDGMENTS

Sequencing of DNA samples was carried out using the equipment of the resource center of the Science Park of St. Petersburg State University “Development of Molecular and Cellular Technologies.”

Molecular dynamics was carried out using the computing resources of the Peter the Great Supercomputer Center of the St. Petersburg Polytechnic University (www.scc.spbstu.ru).

Funding

The study was financially supported by the Russian Foundation for Basic Research (project no. 18-34-00143).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to K. S. Bobrov.

Ethics declarations

COMPLIANCE WITH ETHICAL STANDARDS

This work does not contain any research involving humans or animals as research objects.

Conflict of Interests

The authors declare they have no conflict of interests.

Additional information

Abbreviations: FGly, Cα-formyl glycine; F.p.Sulf-6His, Recombinant Sulfatase from Fusarium proliferatum LE1; pNPS, p-nitrophenyl sulfate; SmCS, sulfatase from Sinorhizobium meliloti; HsS, sulfatase from Homo sapiens; BtS, sulfatase from Bacteroides thetaiotaomicron; MD, molecular dynamics.

Corresponding author: e-mail: bobrov_ks@pnpi.nrcki.ru.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kolchina, N.V., Rychkov, G.N., Kulminskaya, A.A. et al. Structural Organization of the Active Center of Unmodified Recombinant Sulfatase from the Mycelial Fungi Fusarium proliferatum LE1. Russ J Bioorg Chem 46, 563–571 (2020). https://doi.org/10.1134/S1068162020040081

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1068162020040081

Keywords:

  • sulfatase
  • Cα-formylglycine
  • posttranslational modification
  • enzyme-substrate complex