Skip to main content
SpringerLink
Log in

Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH)

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

Abstract

Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.

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

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

Similar content being viewed by others

References

  1. Block RM, Dragun J, Kalinowski TW (1984) Groundwater contamination. 2. Health and enviromental aspects of setting cleanup criteria. Chem Eng 91:70–73

    CAS  Google Scholar 

  2. Dragun J, Kuffner AC, Schneiter RW (1984) Groundwater contamination. 1. Transport and transformation of organic-chemicals. Chem Eng 91:65–70

    CAS  Google Scholar 

  3. Satoh T, Hosokawa M (2000) Organophosphates and their impact on the global environment. Neurotoxicology. 21:223–227

    CAS  PubMed  Google Scholar 

  4. Singh BK, Walker A (2006) Microbial degradation of organophosphorus compounds. FEMS Microbiol Rev 30:428–471

    Article  CAS  PubMed  Google Scholar 

  5. McLoughlin SY, Jackson C, Liu J-W, Ollis DL (2004) Growth of Escherichia coli coexpressing phosphotriesterase and glycerophosphodiester phosphodiesterase, using paraoxon as the sole phosphorus source. Appl Environ Microbiol. 70:404–412

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Harper LL, McDaniel CS, Miller CE, Wild JR (1988) Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes. Appl Environ Microbiol 54:2586–2589

    CAS  PubMed Central  PubMed  Google Scholar 

  7. Yang H, Carr PD, McLoughlin SY, Liu JW, Horne I, Qiu X, Jeffries CMJ, Russell RJ, Oakeshott JG, Ollis DL (2003) Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution. Protein Eng 16:135–145

    Article  CAS  PubMed  Google Scholar 

  8. Sutherland TD, Horne I, Weir KM, Coppin CW, Williams MR, Selleck M, Russell RJ, Oakeshott JG (2004) Enzymatic bioremediation: from the enzyme discovery to applications. Clin Exp Pharmacol Physiol 31:817–821

    Article  CAS  PubMed  Google Scholar 

  9. Ely F, Foo J-L, Jackson CJ, Gahan LR, Ollis DL, Schenk G (2007) Enzymatic bioremediation: organophosphate degradation by binuclear metallo-hydrolases. Curr Top Biochem Res. 9:63–78

    CAS  Google Scholar 

  10. Ely F, Hadler KS, Gahan LR, Guddat LW, Ollis DL, Schenk G (2010) The organophosphate-degrading enzyme from Agrobacterium radiobacter displays mechanistic flexibility for catalysis. Biochem J 432:565–573

    Article  CAS  PubMed  Google Scholar 

  11. Schenk G, Mitić N, Gahan LR, Ollis DL, McGeary RP, Guddat LW (2012) Binuclear metallohydrolases: complex mechanistic strategies for a simple chemical reaction. Acc Chem Res 45:1593–1603

    Article  CAS  PubMed  Google Scholar 

  12. Aubert SD, Li Y, Raushel FM (2004) Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase. Biochemistry 43:5707–5715

    Article  CAS  PubMed  Google Scholar 

  13. Wu F, Li W-S, Chen-Goodspeed M, Sogorb MA, Raushel FM (2000) Rationally engineered mutants of phosphotriesterase for preparative scale isolation of chiral organophosphates. J Am Chem Soc 122:10206–10207

    Article  CAS  Google Scholar 

  14. Hong S-B, Raushel FM (1996) Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase. Biochemistry 35:10904–10912

    Article  CAS  PubMed  Google Scholar 

  15. Raushel FM, Holden HM (2000) Phosphotriesterase: an enzyme in search of its natural substrate. Adv Enzymol Relat Areas Mol Biol 74:51–73

    CAS  PubMed  Google Scholar 

  16. Samples CR, Howard T, Raushel FM, DeRose VJ (2005) Protonation of the binuclear metal center within the active site of phosphotriesterase. Biochemistry 44:11005–11013

    Article  CAS  PubMed  Google Scholar 

  17. Ely F, Hadler KS, Mitic N, Gahan L, Ollis DL, Larrabee JA, Schenk G (2011) Electronic and geometric structure of the organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA). J Biol Inorg Chem 16:777–787

    Article  CAS  PubMed  Google Scholar 

  18. Omburo G, Kuo J, Mullins L, Raushel F (1992) Characterization of the zinc binding site of bacterial phosphotriesterase. J Biol Chem 267:13278–13283

    CAS  PubMed  Google Scholar 

  19. Ely F, Pedroso MM, Gahan LR, Ollis DL, Guddat LW, Schenk G (2012) Phosphate-bound structure of an organophosphate-degrading enzyme from Agrobacterium radiobacter. J Inorg Biochem 106:19–22

    Article  CAS  PubMed  Google Scholar 

  20. Foo J-L, Jackson CJ, Carr PD, Kim H-K, Schenk G, Gahan LR, Ollis DL (2010) Mutation of outer-shell residues modulates metal ion co-ordination strength in a metalloenzyme. Biochem J 429:313–321

    Article  CAS  PubMed  Google Scholar 

  21. MicroCal (2004) ITC data analysis in origin—tutorial guide. MicroCalorimeter User`s Manual, Northampton

    Google Scholar 

  22. Lin LN, Mason AB, Woodworth RC, Brandts JF (1991) Calorimetric studies of the binding of ferric ions to ovotransferrin and interactions between binding sites. Biochemistry 30:11660–11669

    Article  CAS  PubMed  Google Scholar 

  23. Lin LN, Mason AB, Woodworth RC, Brandts JF (1993) Calorimetric studies of the binding of ferric ions to human serum transferrin. Biochemistry 32:9398–9406

    Article  CAS  PubMed  Google Scholar 

  24. Segel IH (1993) Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. Wiley, USA

    Google Scholar 

  25. Thermo Scientific, Grams/AI 9.0 Software (2009)

  26. Shim H, Raushel FM (2000) Self-assembly of the binuclear metal center of phosphotriesterase. Biochemistry 39:7357–7364

    Article  CAS  PubMed  Google Scholar 

  27. Carpenter MC, Wilcox DE (2014) Thermodynamics of formation of the insulin hexamer: metal-stabilized proton-coupled assembly of quaternary structure. Biochemistry 53:1296–1301

    Article  CAS  PubMed  Google Scholar 

  28. Grossoehme NE, Spuches AM, Wilcox DE (2010) Application of isothermal titration calorimetry in bioinorganic chemistry. J Biol Inorg Chem 15:1183–1191

    Article  CAS  PubMed  Google Scholar 

  29. Sigurskjold BW (2000) Exact analysis of competition ligand binding by displacement isothermal titration calorimetry. Anal Biochem 277:260–266

    Article  CAS  PubMed  Google Scholar 

  30. Smith RM, Martell AE, Motekaitis RJ (2004) NIST critically selected stability constants of metal complexes database. Standard Reference Data 46

  31. Adamsky H, Schonherr T, Atanasov M (2004) AOMX: angular overlap model computation. Elsevier, Oxford

    Google Scholar 

  32. Schonherr T, Artanasov M, Adamsky H (2004) AOMX: angular overlap model computation. In: McCleverty JA, Meyer TJ (eds) Comprehensive coordination chemistry II, vol 2. Elsevier, Oxford, pp 443–455

    Chapter  Google Scholar 

  33. Kaden TA, Holmquist B, Vallee BL (1972) Magnetic circular dichroism of cobalt metalloenzyme derivatives. Biochem Biophys Res Commun. 46:1654–1659

    Article  CAS  PubMed  Google Scholar 

  34. Larrabee JA, Leung CH, Moore RL, Thamrong-nawasawat T, Wessler BSH (2004) Magnetic circular dichroism and cobalt(II) binding equilibrium studies of Escherichia coli methionyl aminopeptidase. J Am Chem Soc 126:12316–12324

    Article  CAS  PubMed  Google Scholar 

  35. Larrabee JA, Alessi CM, Asiedu ET, Cook JO, Hoerning KR, Klingler LJ, Okin GS, Santee SG, Volkert TL (1997) Magnetic circular dichroism spectroscopy as a probe of geometric and electronic structure of cobalt(II)-substituted proteins: ground-state zero-field splitting as a coordination number indicator. J Am Chem Soc 119:4182–4196

    Article  CAS  Google Scholar 

  36. Hadler KS, Mitic N, Yip SH-C, Gahan LR, Ollis DL, Schenk G, Larrabee JA (2010) Electronic structure analysis of the dinuclear metal center in the bioremediator glycerophosphodiesterase (GpdQ) from enterobacter aerogenes. Inorg Chem 49:2727–2734

    Article  CAS  PubMed  Google Scholar 

  37. Hadler KS, Tanifum EA, Yip SH-C, Mitic N, Guddat LW, Jackson CJ, Gahan LR, Nguyen K, Carr PD, Ollis DL, Hengge AC, Larrabee JA, Schenk G (2008) Substrate-promoted formation of a catalytically competent binuclear center and regulation of reactivity in a glycerophosphodiesterase from Enterobacter aerogenes. J Am Chem Soc 130:14129–14138

    Article  CAS  PubMed  Google Scholar 

  38. Johansson FB, Bond AD, Nielsen UG, Moubaraki B, Murray KS, Berry KJ, Larrabee JA, McKenzie CJ (2008) Dicobalt II/II, II/III, and III/III complexes as spectroscopic models for dicobalt enzyme active sites. Inorg Chem. 47:5079–5092

    Article  CAS  PubMed  Google Scholar 

  39. Larrabee JA, Chyun S-A, Volwiler AS (2008) Magnetic circular dichroism study of a dicobalt(II) methionine aminopeptidase/fumagillin complex and dicobalt II/II and II/III model complexes. Inorg Chem 47:10499–10508

    Article  CAS  PubMed  Google Scholar 

  40. Hong S-B, Kuo JM, Mullins LS, Raushel FM (1995) CO2 is required for the assembly of the binuclear metal center of phosphotriesterase. J Am Chem Soc 117:7580–7581

    Article  CAS  Google Scholar 

  41. Kiefer LL, Paterno SA, Fierke CA (1995) Hydrogen bond network in the metal binding site of carbonic anhydrase enhances zinc affinity and catalytic efficiency. J Am Chem Soc 117:6831–6837

    Article  CAS  Google Scholar 

  42. Samples CR, Raushel FM, DeRose VJ (2007) Activation of the binuclear metal center through formation of phosphotriesterase; inhibitor complexes. Biochemistry 46:3435–3442

    Article  CAS  PubMed  Google Scholar 

  43. Grimsley JK, Calamini B, Wild JR, Mesecar AD (2005) Structural and mutational studies of organophosphorus hydrolase reveal a cryptic and functional allosteric-binding site. Arch Biochem Biophys 442:169–179

    Article  CAS  PubMed  Google Scholar 

  44. Neri F, Kok D, Miller MA, Smulevich G (1997) Fluoride binding in hemoproteins: the importance of the distal cavity structure. Biochemistry 36:8947–8953

    Article  CAS  PubMed  Google Scholar 

  45. Brändén R, Malmström BG, Vänngård T (1973) The effect of fluoride on the spectral and catalytic properties of the three copper-containing oxidases. Eur J Biochem 36:195

    Article  PubMed  Google Scholar 

  46. Todd MJ, Hausinger RP (2000) Fluoride inhibition of Klebsiella aerogenes urease: mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor. Biochemistry 39:5389–5396

    Article  CAS  PubMed  Google Scholar 

  47. Tormanen CD (2003) Substrate inhibition of rat liver and kidney arginase with fluoride. J Inorg Biochem 93:243

    Article  CAS  PubMed  Google Scholar 

  48. Pethe S, Boucher JL, Mansuy D (2002) Interaction of anions with rat liver arginase: specific inhibitory effects of fluoride. J Inorg Biochem 88:397–402

    Article  CAS  PubMed  Google Scholar 

  49. Cama E, Pethe S, Boucher J-L, Han S, Emig FA, Ash DE, Viola RE, Mansuy D, Christianson DW (2004) Inhibitor coordination interactions in the binuclear manganese cluster of arginase. Biochemistry 43:8987–8999

    Article  CAS  PubMed  Google Scholar 

  50. Mitić N, Valizadeh M, Leung EWW, de Jersey J, Hamilton S, Hume DA, Cassady AI, Schenk G (2005) Human tartrate-resistant acid phosphatase becomes an effective ATPase upon proteolytic activation. Arch Biochem Biophys 439:154–164

    Article  PubMed  Google Scholar 

  51. Merkx M, Pinkse MWH, Averill BA (1999) Evidence for nonbridged coordination of p-nitrophenyl phosphate to the dinuclear Fe(III), Mn(II) center in bovine spleen purple acid phosphatase during enzymatic turnover. Biochemistry 38:9914–9925

    Article  CAS  PubMed  Google Scholar 

  52. Elliott TW, Mitic N, Gahan LR, Guddat LW, Schenk G (2006) Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism. J Braz Chem Soc 17:1558–1565

    Article  CAS  Google Scholar 

  53. Wang X, Ho RYN, Whiting AK, Que L (1999) Spectroscopic characterization of a ternary phosphatase, substrate, fluoride complex. Mechanistic implications for dinuclear hydrolases. J Am Chem Soc. 121:9235–9236

    Article  CAS  Google Scholar 

  54. Schenk G, Elliott TW, Leung E, Carrington LE, Mitić N, Gahan LR, Guddat LW (2008) Crystal structures of a purple acid phosphatase, representing different steps of this enzyme’s catalytic cycle. BMC Struct Biol 8:6

    Article  PubMed Central  PubMed  Google Scholar 

  55. Jackson C, Kim H-K, Carr PD, Liu J-W, Ollis DL (2005) The structure of an enzyme-product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism. Biochimica et Biophysica Acta (BBA) Proteins Proteomics 1752:56–64

    Article  CAS  Google Scholar 

  56. Benning MM, Shim H, Raushel FM, Holden HM (2001) High resolution X-ray structures of different metal-substituted forms of phosphotriesterase from Pseudomonas diminuta. Biochemistry 40:2712–2722

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work is financially supported by the Australian Research Council, Discovery Projects Scheme (DP120104263). G. S. also acknowledges the receipt of an ARC Future Fellowship (FT120100694). M. P. is supported by an International Postgraduate Research Scholarship and University of Queensland International Living Allowance Scholarship. J. A. L. and D. E. W. thank the National Science Foundation (USA) for financial support from grants CHE0848433, CHE1303852, and CHE0820965 (MCD instrument) to J. A. L. and CHE1308598 to D. E. W. N. M. would like to thank the Science Foundation Ireland for financial support in form of a President of Ireland Young Researcher Award (SFI-PIYRA).

Author information

Authors and Affiliations

  1. School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD, 4072, Australia

    Marcelo M. Pedroso, Fernanda Ely, Lawrence R. Gahan & Gerhard Schenk

  2. Department of Chemistry, National University of Ireland, Maynooth, Maynooth, Co. Kildare, Ireland

    Nataša Mitić

  3. Department of Chemistry, Dartmouth College, Hanover, NH, 03755, USA

    Margaret C. Carpenter & Dean E. Wilcox

  4. Department of Chemistry and Biochemistry, Middlebury College, Middlebury, VT, 05753, USA

    James L. Larrabee

  5. Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia

    David L. Ollis

Authors
  1. Marcelo M. Pedroso
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fernanda Ely
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nataša Mitić
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Margaret C. Carpenter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lawrence R. Gahan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dean E. Wilcox
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. James L. Larrabee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David L. Ollis
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gerhard Schenk
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gerhard Schenk.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 286 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pedroso, M.M., Ely, F., Mitić, N. et al. Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH). J Biol Inorg Chem 19, 1263–1275 (2014). https://doi.org/10.1007/s00775-014-1183-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-014-1183-9

Keywords

Search

Navigation