Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH)
- 539 Downloads
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.
KeywordsBinding affinity Calorimetry Enzyme kinetics Magnetic circular dichroism Site-directed mutagenesis
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).
- 1.Block RM, Dragun J, Kalinowski TW (1984) Groundwater contamination. 2. Health and enviromental aspects of setting cleanup criteria. Chem Eng 91:70–73Google Scholar
- 2.Dragun J, Kuffner AC, Schneiter RW (1984) Groundwater contamination. 1. Transport and transformation of organic-chemicals. Chem Eng 91:65–70Google 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. 9:63–78Google Scholar
- 21.MicroCal (2004) ITC data analysis in origin—tutorial guide. MicroCalorimeter User`s Manual, NorthamptonGoogle Scholar
- 24.Segel IH (1993) Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. Wiley, USAGoogle Scholar
- 25.Thermo Scientific, Grams/AI 9.0 Software (2009)Google Scholar
- 30.Smith RM, Martell AE, Motekaitis RJ (2004) NIST critically selected stability constants of metal complexes database. Standard Reference Data 46Google Scholar
- 31.Adamsky H, Schonherr T, Atanasov M (2004) AOMX: angular overlap model computation. Elsevier, OxfordGoogle 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 119:4182–4196CrossRefGoogle 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 130:14129–14138PubMedCrossRefGoogle Scholar