Skip to main content
SpringerLink
Log in

Fluoride inhibition of Sporosarcina pasteurii urease: structure and thermodynamics

  • Original Paper
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

Urease is a nickel-dependent enzyme and a virulence factor for ureolytic bacterial human pathogens, but it is also necessary to convert urea, the most worldwide used fertilizer, into forms of nitrogen that can be taken up by crop plants. A strategy to control the activity of urease for medical and agricultural applications is to use enzyme inhibitors. Fluoride is a known urease inhibitor, but the structural basis of its mode of inhibition is still undetermined. Here, kinetic studies on the fluoride-induced inhibition of urease from Sporosarcina pasteurii, a widespread and highly ureolytic soil bacterium, were performed using isothermal titration calorimetry and revealed a mixed competitive and uncompetitive mechanism. The pH dependence of the inhibition constants, investigated in the 6.5–8.0 range, reveals a predominant uncompetitive mechanism that increases by increasing the pH, and a lesser competitive inhibition that increases by lowering the pH. Ten crystal structures of the enzyme were independently determined using five crystals of the native form and five crystals of the protein crystallized in the presence of fluoride. The analysis of these structures revealed the presence of two fluoride anions coordinated to the Ni(II) ions in the active site, in terminal and bridging positions. The present study consistently supports an interaction of fluoride with the nickel centers in the urease active site in which one fluoride competitively binds to the Ni(II) ion proposed to coordinate urea in the initial step of the catalytic mechanism, while another fluoride uncompetitively substitutes the Ni(II)-bridging hydroxide, blocking its nucleophilic attack on urea.

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

Scheme 1
Scheme 2
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Scheme 3
Scheme 4

Similar content being viewed by others

References

  1. Maroney MJ, Ciurli S (2014) Chem Rev 114:4206–4228

    Article  CAS  PubMed  Google Scholar 

  2. Mobley HLT, Hausinger RP (1989) Microbiol Rev 53:85–108

    CAS  PubMed Central  PubMed  Google Scholar 

  3. Karplus PA, Pearson MA, Hausinger RP (1997) Acc Chem Res 30:330–337

    Article  CAS  Google Scholar 

  4. Krajewska B (2009) J Mol Cat B Enzymatic 59:9–21

    Article  CAS  Google Scholar 

  5. Zambelli B, Musiani F, Benini S, Ciurli S (2011) Acc Chem Res 44:520–530

    Article  CAS  PubMed  Google Scholar 

  6. Callahan BP, Yuan Y, Wolfenden R (2005) J Am Chem Soc 127:10828–10829

    Article  CAS  PubMed  Google Scholar 

  7. Ciurli S, Benini S, Rypniewski WR, Wilson KS, Miletti S, Mangani S (1999) Coord Chem Rev 190–192:331–355

    Article  Google Scholar 

  8. Burne RA, Chen YY (2000) Microbes Infect 2:533–542

    Article  CAS  PubMed  Google Scholar 

  9. Ciurli S (2007) Nickel and its surprising impact in nature. Wiley, Chichester, pp 241–278

    Book  Google Scholar 

  10. Benini S, Musiani F, Ciurli S (2013) In: Kretsinger RH, Uversky VN, Permyakov EA (eds) Encyclopedia of metalloproteins. Springer, New York, pp 2287–2292

    Chapter  Google Scholar 

  11. Farrugia MA, Macomber L, Hausinger RP (2013) J Biol Chem 288:13178–13185

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Jabri E, Carr MB, Hausinger RP, Karplus PA (1995) Science 268:998–1004

    Article  CAS  PubMed  Google Scholar 

  13. Benini S, Rypniewski WR, Wilson KS, Miletti S, Ciurli S, Mangani S (1999) Structure 7:205–216

    Article  CAS  PubMed  Google Scholar 

  14. Ha N-C, Oh S-T, Sung JY, Cha KA, Lee MH, Oh B-H (2001) Nat Struct Biol 8:505–509

    Article  CAS  PubMed  Google Scholar 

  15. Balasubramanian A, Ponnuraj K (2010) J Mol Biol 400:274–283

    Article  CAS  PubMed  Google Scholar 

  16. Balasubramanian A, Durairajpandian V, Elumalai S, Mathivanan N, Munirajan AK, Ponnuraj K (2013) Int J Biol Macromol 58:301–309

    Article  CAS  PubMed  Google Scholar 

  17. Kosikowska P, Berlicki Ł (2011) Expert Opin Ther Pat 21:945–957

    Article  CAS  PubMed  Google Scholar 

  18. Vassiliou S, Grabowiecka A, Kosikowska P, Yiotakis A, Kafarski P, Berlicki Ł (2008) J Med Chem 51:5736–5744

    Article  CAS  PubMed  Google Scholar 

  19. Vassiliou S, Kosikowska P, Grabowiecka A, Yiotakis A, Kafarski P, Berlicki Ł (2010) J Med Chem 53:5597–5606

    Article  CAS  PubMed  Google Scholar 

  20. Berlicki Ł, Bochno M, Grabowiecka A, Białas A, Kosikowska P, Kafarski P (2012) Amino Acids 42:1937–1945

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Benini S, Rypniewski WR, Wilson KS, Ciurli S, Mangani S (1998) J Biol Inorg Chem 3:268–273

    Article  CAS  Google Scholar 

  22. Pearson MA, Overbye Michel L, Hausinger RP, Karplus PA (1997) Biochemistry 36:8164–8172

    Article  CAS  PubMed  Google Scholar 

  23. Benini S, Rypniewski WR, Wilson KS, Miletti S, Ciurli S, Mangani S (2000) J Biol Inorg Chem 5:110–118

    Article  CAS  PubMed  Google Scholar 

  24. Benini S, Rypniewski WR, Wilson KS, Ciurli S, Mangani S (2001) J Biol Inorg Chem 6:778–790

    Article  CAS  PubMed  Google Scholar 

  25. Benini S, Rypniewski WR, Wilson KS, Mangani S, Ciurli S (2004) J Am Chem Soc 126:3714–3715

    Article  CAS  PubMed  Google Scholar 

  26. Benini S, Kosikowska P, Cianci M, Mazzei L, Vara AG, Berlicki Ł, Ciurli S (2013) J Biol Inorg Chem 18:391–399

    Article  CAS  PubMed  Google Scholar 

  27. Dixon NE, Blakeley RL, Zerner B (1980) Can J Biochem 58:481–488

    Article  CAS  PubMed  Google Scholar 

  28. Todd MJ, Hausinger RP (2000) Biochemistry 39:5389–5396

    Article  CAS  PubMed  Google Scholar 

  29. Saboury AA, Moosavi-Movahedi AA (1997) J Enz Inhib 12:273–279

    Article  CAS  Google Scholar 

  30. Krajewska B, Zaborska W, Leszko M (2001) J Mol Catal B 14:101–109

    Article  CAS  Google Scholar 

  31. Todd MJ, Gomez J (2001) Anal Biochem 296:179–187

    Article  CAS  PubMed  Google Scholar 

  32. Mazzei L, Ciurli S, Zambelli B (2014) J Vis Exp (86):e51487. doi:10.3791/51487

  33. Segel IH (1993) Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. Wiley-Interscience, New York

    Google Scholar 

  34. Kabsch W (2010) Acta Crystallogr D Biol Crystallogr 66:125–132

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. N. Collaborative Computational Project (1994) Acta Cryst D50:760–763

    Google Scholar 

  36. Murshudov GN, Vagin AA, Dodson EJ (1997) Acta Crystallogr D Biol Crystallogr 53:240–255

    Article  CAS  PubMed  Google Scholar 

  37. Murshudov GN, Vagin AA, Lebedev A, Wilson KS, Dodson EJ (1999) Acta Crystallogr D Biol Crystallogr 55:247–255

    Article  CAS  PubMed  Google Scholar 

  38. Emsley P, Cowtan K (2004) Acta Crystallogr D Biol Crystallogr 60:2126–2132

    Article  PubMed  Google Scholar 

  39. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Acta Crystallogr D Biol Crystallogr 66:486–501

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) J Comput Chem 25:1605–1612

    Article  CAS  PubMed  Google Scholar 

  41. Pedroso MM, Ely F, Lonhienne T, Gahan LR, Ollis DL, Guddat LW, Schenk GG (2014) J Biol Inorg Chem 19:389–398

    Article  CAS  PubMed  Google Scholar 

  42. Ciurli S, Marzadori C, Benini S, Deiana S, Gessa C (1996) Soil Biol Biochem 28:811–817

    Article  CAS  Google Scholar 

  43. Benini S, Gessa C, Ciurli S (1996) Soil Biol Biochem 28:819–821

    Article  CAS  Google Scholar 

  44. Quiroz-Valenzuela S, Sukuru SC, Hausinger RP, Kuhn LA, Heller WT (2008) Arch Biochem Biophys 480:51–57

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Cruickshank DWJ (1999) Acta Cryst D55:583–601

    CAS  Google Scholar 

  46. Addison AW, Rao TN, Reedijk J, van Rijn J, Verschoor GC (1984) J Chem Soc Dalton Trans (7):1349–1356

  47. Mystkowski EM (1928) Acta Biol Exp. Varsovie 2:212–224

    Google Scholar 

  48. Pearson RM, Smith J (1943) Biochem J 37:153–164

  49. Dixon NE, Gazzola C, Blakeley R, Zerner B (1975) J Am Chem Soc 97:4131–4132

    Article  CAS  PubMed  Google Scholar 

  50. Samygina VR, Moiseev VM, Rodina EV, Vorobyeva NN, Popov AN, Kurilova SA, Nazarova TI, Avaeva SM, Bartunik HD (2007) J Mol Biol 366:1305–1317

    Article  CAS  PubMed  Google Scholar 

  51. Cama E, Pethe S, Boucher JL, Han S, Emig FA, Ash DE, Viola RE, Mansuy D, Christianson DW (2004) Biochemistry 43:8987–8999

    Article  CAS  PubMed  Google Scholar 

  52. Qin J, Chai G, Brewer JM, Lovelace LL, Lebioda L (2006) Biochemistry 45:793–800

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. Thorsell A-G, Persson C, Grslund S, Hammarstrm M, Busam RD, Hallberg BM (2009) Proteins Struct Funct Bioinform 77:242–246

    Article  CAS  Google Scholar 

  54. Schenk G, Elliott TW, Leung E, Carrington LE, Mitic N, Gahan LR, Guddat LW (2008) BMC Struct Biol 8:6

    Article  PubMed Central  PubMed  Google Scholar 

  55. Musiani F, Arnofi E, Casadio R, Ciurli S (2001) J Biol Inorg Chem 6:300–314

    Article  CAS  PubMed  Google Scholar 

  56. Christianson DW (2005) Acc Chem Res 38:191–201

    Article  CAS  PubMed  Google Scholar 

  57. Mitić NN, Smith SJS, Neves AA, Guddat LW, Gahan LRL, Schenk GG (2006) Chem Rev 106:3338–3363

    Article  PubMed  Google Scholar 

  58. Schenk GG, Mitić NN, Gahan LR, Ollis DL, McGeary RP, Guddat LW (2012) Acc Chem Res 45:1593–1603

    Article  CAS  PubMed  Google Scholar 

  59. Pethe SS, Boucher JLJ, Mansuy DD (2002) J Inorg Biochem 88:397–402

    Article  CAS  PubMed  Google Scholar 

  60. Xie X-Y, Wang C-X, Wang Z-Y (2004) J Therm Anal Calorim 77:1005–1012

    Article  CAS  Google Scholar 

  61. Elliot TW, Mitić NN, Gahan LR, Guddat LW, Schenk GG (2006) J Braz Chem Soc 17:1558–1565

    Article  Google Scholar 

  62. Mitić NN, Valizadeh M, Leung EW, de Jersey J, Hamilton S, Hume DA, Cassady AI, Schenk GG (2005) Arch Biochem Biophys 439:154–164

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

Dr. Barbara Zambelli is acknowledged for useful discussions. Data were collected under project MX-40 at EMBL MX-P13 beamline at Petra III, c/o DESY (Hamburg, DE). LM was supported by a fellowship financed by Specialty Fertilizer Products (Leawood, KS, USA) and by a Ph.D. fellowship funded by the University of Bologna. The research is partially funded by CIRMMP (Consorzio Interuniversitario di Risonanze Magnetiche di Metallo-Proteine).

Author information

Authors and Affiliations

  1. Faculty of Science and Technology, Free University of Bolzano, Piazza Università 5, 39100, Bolzano, Italy

    Stefano Benini

  2. European Molecular Biology Laboratory c/o DESY, Notkestrasse 85, 22603, Hamburg, Germany

    Michele Cianci

  3. Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Via Fanin 40, 40127, Bologna, Italy

    Luca Mazzei & Stefano Ciurli

Authors
  1. Stefano Benini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michele Cianci
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Luca Mazzei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stefano Ciurli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Stefano Benini or Stefano Ciurli.

Additional information

An Interactive 3D Complement page in Proteopedia is available at: http://proteopedia.org/wiki/index.php/Journal:JBIC:26.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Benini, S., Cianci, M., Mazzei, L. et al. Fluoride inhibition of Sporosarcina pasteurii urease: structure and thermodynamics. J Biol Inorg Chem 19, 1243–1261 (2014). https://doi.org/10.1007/s00775-014-1182-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-014-1182-x

Keywords

Search

Navigation