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JBIC Journal of Biological Inorganic Chemistry

, Volume 19, Issue 8, pp 1263–1275 | Cite as

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

  • Marcelo M. Pedroso
  • Fernanda Ely
  • Nataša Mitić
  • Margaret C. Carpenter
  • Lawrence R. Gahan
  • Dean E. Wilcox
  • James L. Larrabee
  • David L. Ollis
  • Gerhard Schenk
Original Paper

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.

Keywords

Binding affinity Calorimetry Enzyme kinetics Magnetic circular dichroism Site-directed mutagenesis 

Notes

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).

Supplementary material

775_2014_1183_MOESM1_ESM.pdf (286 kb)
Supplementary material 1 (PDF 286 kb)

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Copyright information

© SBIC 2014

Authors and Affiliations

  • Marcelo M. Pedroso
    • 1
  • Fernanda Ely
    • 1
  • Nataša Mitić
    • 2
  • Margaret C. Carpenter
    • 3
  • Lawrence R. Gahan
    • 1
  • Dean E. Wilcox
    • 3
  • James L. Larrabee
    • 4
  • David L. Ollis
    • 5
  • Gerhard Schenk
    • 1
  1. 1.School of Chemistry and Molecular BiosciencesThe University of QueenslandSt. LuciaAustralia
  2. 2.Department of ChemistryNational University of Ireland, MaynoothCo. KildareIreland
  3. 3.Department of ChemistryDartmouth CollegeHanoverUSA
  4. 4.Department of Chemistry and BiochemistryMiddlebury CollegeMiddleburyUSA
  5. 5.Research School of ChemistryAustralian National UniversityCanberraAustralia

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