Skip to main content
SpringerLink
Log in

Active Site Modifications of Organophosphorus Hydrolase for Improved Detoxification of Organophosphorus Neurotoxins

  • Chapter
Enzymes in Action

Part of the book series: NATO Science Partnership Sub-Series: 1: ((ASDT,volume 33))

Abstract

Amino acid substitutions within the active site of the dimeric metalloenzyme Organophosphorus Hydrolase (OPH) result in a striking enhancement in the hydrolysis of certain chemical warfare agents and their analogues. These changes alter the metal content of the enzyme and we suggest that changes in metal requirements improve the catalytic characteristics by allowing greater structural flexibility and access of larger substrates to the active site. Crystallographic and three-dimensional modeling analyses have suggested that removing steric hindrances in the vicinity of the binding pocket could further enhance the effectiveness of OPH to hydrolyze VX. These studies also suggest that the hydrogen-bonding network supplying support and stability to the active site deserve a critical analysis for further catalytic improvements. The broad substrate specificity and hydrolytic efficiency of OPH and the ability to genetically engineer the enzyme for specific target organophosphate neurotoxins have provided realistic OPHbased technologies for detoxification of these compounds, including enzyme immobilization on various matrices, discriminative detection, and clinical therapy. A series of optimized enzymes for individual substrates can be envisioned that would maximize degradative activity under a particular environmental situation. The capacity for further improvement is remarkable, and the opportunity for a variety of biotechnological applications is quite pronounced.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
eBook
USD 16.99
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 54.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Barrett, D.S. and F.W. Oehme. (1985) A review of organophosphorus ester-induced delayed neurotoxicityVet.and Hum. Toxicol.2722–37.

    CAS  Google Scholar 

  2. Johnson, M.K. (1975) The delayed neuropathy caused by some organophosphorus esters: mechanism and challengeCRC Crit. Rev. Toxicol.3289–316.

    Article  CAS  Google Scholar 

  3. Johnson, M.K. (1987) Receptor or enzyme: The puzzle of NTE and organophosphate-induced delayed polyneuropathyTrends Pharmacol8, 174–179.

    Article  CAS  Google Scholar 

  4. Cavanagh, J.B. (1954) The toxic effects of tri-ortho-cresyl phosphate on the nervous system, and experimental study in hensJ. Neurol. Neurosurg. Psychiat.17163–172.

    Article  CAS  Google Scholar 

  5. Sprague, G.L. and Bickford, A.A. (1981) Effect of multiple diisopropylfluorophosphate injections in hens: behavioral, biochemical and histological investigationJ. Toxicol. Environ. Health 8973–988.

    Article  CAS  Google Scholar 

  6. Chae, M.Y., Postula, J.F., and Raushel, F.M. (1994) Stereospecific enzymatic hydrolysis of phosphorus-sulfur bonds in chiral organophosphate triestersBioorg. Med. Chem. Lett.41473–1478.

    Article  CAS  Google Scholar 

  7. Lai, K., Stolowich, N.J., and Wild, J.R. (1995) Characterization of P-S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolaseArch. Biochem. Biophys 31859–64.

    Article  CAS  Google Scholar 

  8. Kolakowski, J.E., DeFrank, J.J., Harvey, S.P., Szafraniec, L.L., Beaudry, W.T., Lai, K. and Wild, J.R. (1997) Enzymatic hydrolysis of the chemical warfare agent VX and its neurotoxic analogues by organophosphorus hydrolaseBiocatal. Biotransform.15297–312.

    Article  CAS  Google Scholar 

  9. Dumas, D.P., Durst, H. D., Landis, W.G., Raushel, F.M., and Wild, J.R. (1990) Inactivation of organophosphorus nerve agents by the phosphotriesterase fromPseudomonas diminuta Arch. Biochem. Biophys.277155–159.

    Article  CAS  Google Scholar 

  10. DeFrank, J.J., Beaudry, W.T., Cheng, T.-C., Harvey, S.P., Stroup, A.N., and Szafraniec, L.L. (1993) Screening of halophilic bacteria andAlteromonasspecies for organophosphorus hydrolyzing enzyme activityChem.-Biol. Interact.87141–148.

    Article  CAS  Google Scholar 

  11. Cheng, T.-C., Harvey, S.P., and Chen, G.L. (1996) Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzymeAppl. Environ. Microbio!.621636–1641.

    CAS  Google Scholar 

  12. Hassett, C., Richter, R.J., Humbert, R., Chapline, C., Crabb, J.W., Omiecinski, C.J., and Furlong, C.E. (1991) Characterization of cDNA clones encoding rabbit and human serum paraoxonase: the mature protein retains its signal sequenceBiochemistry 3010141–10149.

    Article  CAS  Google Scholar 

  13. Hoskin, F.C.G. and Roush, A.H. (1982) Hydrolysis of nerve gas by squid-type diisopropyl phosphorofluoridate hydrolyzing enzyme on agarose resinScience 2151255–1257.

    Article  CAS  Google Scholar 

  14. Mulbry, W.W., Karns, J.S., Keamey, P.C., Nelson, J.O., McDaniel, C.S., and Wild, J. R. (1986) Identification of a plasmid-borne parathion hydrolase gene fromFlavobacteriumsp. by Southern hybridization withopdfromPseudomonas diminuta Appl. Environ. Microbiol.51926–930.

    CAS  Google Scholar 

  15. Harper, L.L., McDaniel, C.S. Miller, C.E., and Wild, J.R. (1988) Dissimilar plasmids isolated fromPseudomonas diminutaMG anda Flavobacteriumsp. (ATCC 27551) contain identicalopdgenesAppl. Environ. Microbio!.542586–2589.

    CAS  Google Scholar 

  16. McDaniel, C.S., Harper, L.L., and Wild, J. R. (1988) Cloning and sequencing of a plasmid-borne gene(opd)encoding a phosphotriesteraseJ. Bacteriol.1702306–2311.

    CAS  Google Scholar 

  17. Mulbry, W.W. and Karns, J.S. (1989) Parathion hydrolase specified by theFlavobacterium opdgene: relationship between the gene and the proteinJ. Bacteriol.1716740–6746.

    CAS  Google Scholar 

  18. Serdar, C.M., Murdock, D.C., and Rohde, M.F. (1989) Parathion hydrolase gene fromPseudomonas diminutaMG: subcloning, complete nucleotide sequence, and expression of the mature portion of the enzyme inEscherichia coli Bio/Fechnol.71151–1155.

    CAS  Google Scholar 

  19. Steiert, J.G., Pogell, B.M., Speedie, M.K., and Laredo, J. (1989) A gene coding for a membrane-bound hydrolase is expressed as a secreted, soluble enzyme inStreptomyces lividans Bio/I’echnol.765–68.

    Article  CAS  Google Scholar 

  20. Phillips, J.P., Xin, J.H., Kirby, K., Milne, J. C. P., Krell, P., and Wild, J.R. (1990) Transfer and expression of an organophosphate insecticide-degrading gene fromPseudomonas in Drosophila melanogaster Proc. Natl. Acad. Sci. U. S. A.878155–8159.

    Article  CAS  Google Scholar 

  21. Dave, K.I., Lauriano, C., Xu, B., Wild, J.R., and Kenerley, C.M. (1994) Expression of organophosphate hydrolase in the filamentous fungusGliocladium virens Appl. Microbio!. Biotechnol.41352–358.

    Article  CAS  Google Scholar 

  22. Dumas, D.P., Caldwell, S.R., Wild, J.R., and Raushel, F.M. (1989) Purification and properties of the phosphotriesterase fromPseudomonas diminuta J. Biol. Chem.26419659–19665.

    CAS  Google Scholar 

  23. Hoskin, F.C.G., Walker, J.E., Dettbarn, W.-D., and Wild, J.R. (1995) Hydrolysis of tetriso by an enzyme derived fromPseudomonas diminuta asa model for the detoxication of 0-ethyl S-(2diisopropylaminoethyl) methylphosphonothiolate (VX)Biochem. Phatrnacol.49711–715.

    Article  CAS  Google Scholar 

  24. Benning, M.W., Kuo, J.M., Raushel, F.M., and Holden, H.M. (1994) Three-dimensional structure of Phosphotriesterase: An enzyme capable of detoxifying organophosphate nerve agentsBiochemistry33, 15001–15007.

    Article  CAS  Google Scholar 

  25. Benning, M.W., Kuo, J.M., Raushel, F.M., and Holden, H.M. (1995) Three-dimensional structure of the binuclear metal center of PhosphotriesteraseBiochemistry 347973–7978.

    Article  CAS  Google Scholar 

  26. Vanhooke, J.L., Benning, M.M., Raushel, F.M., and Holden, H.M. (1996) Three-dimensional structure of the zinc-containing Phosphotriesterase with the bound substrate analog diethyl 4methylbenzylphosphonateBiochemistry 35 6020–6025.

    Article  CAS  Google Scholar 

  27. Omburo, G.A., Kuo, J.M., Mullins, L.S., and Raushel, F.M. (1992) Characterization of the zinc binding site of bacterial phosphotriesteraseJ. Biol. Chem.26713278–13283.

    CAS  Google Scholar 

  28. Lai, K., Dave, K.I., and Wild, J.R. (1994) Enzymatic decontamination of organophosphorus chemical agents by genetic and biochemical manipulation of organophosphorus hydrolase, in D. A. Berg, J. D. Williams, Jr., and P. J. Reeves (eds.)Proceedings of the 1993 ERDEC Scientific Conference on Chemical Defense Ressearch 16–19 November 1993U.S. Army, Aberdeen Proving Ground, MD, pp.887–894.

    Google Scholar 

  29. Grimsley, I.K., Scholtz, J.M., Pace, C.N., and Wild, J.R. (1997) Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimericintermediate Biochemistry 3614366–14374.

    Article  CAS  Google Scholar 

  30. Lai, K., Dave, K.I., and Wild, J.R. (1994) Bimetallic binding motifs in organophosphorus hydrolase are important for catalysis and structural organizationJ. Biol. Chem.26916579–16584.

    CAS  Google Scholar 

  31. Kuo, J.M., and Raushel, F.M. (1994) Identification of the histidine ligands to the binuclear metal center of phosphotriesterase by site-directed mutagenesisBiochemistry33, 4265–4272.

    Article  CAS  Google Scholar 

  32. Lai, K. Modification and characterization of the neurotoxic substrate specificity of organophosphorus hydrolase, Ph.D. Dissertation, Texas A&M University, 1994.

    Google Scholar 

  33. Kuo, J.M. Investigations of the active site and metal centers of phosphotriesterase, Ph.D. Dissertation, Texas A&M University, 1995.

    Google Scholar 

  34. diSioudi, B.,Grimsley, J.K., Lai, K., and Wild, J.R. (1999) Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificityBiochemistry 382866–2872.

    Article  CAS  Google Scholar 

  35. Lewis, V.E., Donarski, W.J., Wild, J.R., and Raushel, F.M. (1988) Mechanism and stereochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesteraseBiochemistry 271591–1597.

    Article  CAS  Google Scholar 

  36. Watkins, L.M., Mahoney, H.J., McCulloch, J.K., and Raushel, F.M. (1997) Augmented hydrolysis of diisopropyl fluorophosphate in engineered mutants of phosphotriesteraseJ. Biol. Chem.27225596–25601.

    Article  CAS  Google Scholar 

  37. Rainina, E.I., Efremenco, E.N., and Varfolomeyev, S.D. (1996) The development of a new biosensor based on recombinantE. colsfor the direct detection of organophosphorus neurotoxinsBiosens. Bioelectron.11 991–1000.

    Article  CAS  Google Scholar 

  38. Simonian, A. L., Rainina, E.I., and Wild, J.R. (1997) A new approach for discriminative detection of organophosphate neurotoxins in the presence of other cholinesterase inhibitorsAnal. Lett.30, 2453–2468.

    Article  CAS  Google Scholar 

  39. Mulchandani, A., Mulchandani, P., Kaneve, I., and Chen, W. (1998) Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 1. Potentiometric microbial electrodeAnal. Chem.704140–4145.

    Article  CAS  Google Scholar 

  40. Mulchandani, A., Kaneve, I., and Chen, W. (1998) Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 2. Fiber-optic microbial biosensorAnal. Chem.705042–5046.

    Article  CAS  Google Scholar 

  41. Yang, F., Wild, J.R., and Russell, A.J. (1995) Nonaqueous biocatalytic degradation of a nerve gasmimic Biotechnol. Prog.11471–474.

    Article  CAS  Google Scholar 

  42. LeJenne, K.E. and Russell, A.J. (1996) Covalent binding of a nerve agent hydrolyzing enzyme within polyurethane foamsBiotechnol. Bioeng.51450–457.

    Article  Google Scholar 

  43. LeJeune, K.E., Mesiano, A.J., Bower, S.B., Grimsley, J.K., Wild, J.R., and Russell, A.J. (1997) Dramatically stabilized phosphotriesterase-polymers for nerve agent degradationBiotechnol. Bioeng.54105–114.

    Article  CAS  Google Scholar 

  44. LeJeune, K.E., Wild, J.R., and Russell, A.J. (1998) Nerve agents degraded by enzymatic foamsNature 39527–28.

    Article  CAS  Google Scholar 

  45. Ecobichon, D.J. (1991) Toxic effects of pesticides, in: M.O. Amdur, J. Doull, and C.D. Klaassen, (eds.)Casarett and Doull’s Toxicology: The Basic Science of PoisonsMcGraw-Hill, New York, pp. 565–622.

    Google Scholar 

  46. Matsumura, F. (1985) Toxicology of Insecticides, Plenum Press, New York.

    Google Scholar 

  47. World Health Organization (WHO) in collaboration with United Nations Environment Programme, Public Health Impact of Pesticides Used in Agriculture, (1990) World Health Organization, Geneva, p.128.

    Google Scholar 

  48. Ashani,Y., Rothschild, N., Segall, Y., Lecanon, D., and Raveh, L. (1991) Prophylaxis against organophosphate poisoning by an enzyme hydrolysing organophosphorus compounds in miceLife Sci.49367–374.

    Article  CAS  Google Scholar 

  49. Kaliste-Korhonen, E., Ylitalo, P., Hänninen, O., and Raushel, F.M. (1993) Phosphotriesterase decreases paraoxon toxicity in miceToxicol. Appl. Pharmacol.121275–278.

    Article  CAS  Google Scholar 

  50. Tuovinen, K., Kaliste-Korhonen, E., Raushel, F.M., and Hänninen, O. (1994) Phosphotriesterase — a promising candidate for use in detoxification of organophosphatesFundam. Appl. Toxicol.23578–584.

    Article  CAS  Google Scholar 

  51. Tuovinen, K., Kaliste-Korhonen, E., Raushel, F.M., and Hänninen, O. (1996) Protection of organophosphate-inactivated esterases with phosphotriesteraseFundam. Appl. Toxicol.31210–217.

    Article  CAS  Google Scholar 

  52. Tuovinen, K., Kaliste-Korhonen, E., Raushel, F.M.,and Hänninen, O. (1999) Success of pyridostigmine, physostigmine, eptastigmine and phosphotriesterase treatments in acute sann intoxicationToxicology 134169–178.

    CAS  Google Scholar 

  53. Pei, L., Omburo, G., McGuinn, W.D., Petrikovics, I., Dave, K., Raushel, F.M., Wild, J.R., DeLoach, J.R., and Way, J.L. (1994) Encapsulation of phosphotriesterase within murine erythrocytesToxicol. Appl. Pharmacol.124296–301.

    Article  CAS  Google Scholar 

  54. Pei, L. Petrikovics, I., and Way, J.L. (1995) Antagonism of the lethal effects of paraoxon by carrier erythrocytes containing phosphotriesteraseFundam. Appl. Toxicol.28209–214.

    CAS  Google Scholar 

  55. Petrikovics, I., Hong, K., Omburo, G., Hu, QZ., Pei, L., McGuinn, W.D., Sylvester, D., Tamulinas, C., Papahadjopoulos, D., Jaszberenyi, J.C., and Way, J.L. (1999) Antagonism of paraoxon intoxication by recombinant phosphotriesterase encapsulated within sterically stabilized liposomesToxicol. Appl. Phannacol.15656–63.

    Article  CAS  Google Scholar 

  56. Masson, P., Josse, D., Lockridge, O., Viguie, N., Taupin, C., and Buhler, C. (1998) Enzymes hydrolyzing organophosphates as potential catalytic scavengers against organophosphate poisoningJ. Physiology (Paris) 92357–362.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, 77843-2128, USA

    Janet K. Grimsley, Thomas R. Holton, James C. Sacchettini & James R. Wild

  2. Scott Foresman, Educational Technology, 1900 East Lake Avenue, Glenview, IL, 60025

    Barbara D. Disioudi

Authors
  1. Janet K. Grimsley
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Barbara D. Disioudi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas R. Holton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. James C. Sacchettini
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. James R. Wild
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Organic Chemistry, NSR Institute for Molecular Structure, Design and Synthesis, University of Nijmegen, The Netherlands

    Binne Zwanenburg

  2. Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Łódź, Poland

    Marian Mikołajczyk  & Piotr Kiełbasiński  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2000 Springer Science+Business Media Dordrecht

About this chapter

Cite this chapter

Grimsley, J.K., Disioudi, B.D., Holton, T.R., Sacchettini, J.C., Wild, J.R. (2000). Active Site Modifications of Organophosphorus Hydrolase for Improved Detoxification of Organophosphorus Neurotoxins. In: Zwanenburg, B., Mikołajczyk, M., Kiełbasiński, P. (eds) Enzymes in Action. NATO Science Partnership Sub-Series: 1:, vol 33. Springer, Dordrecht. https://doi.org/10.1007/978-94-010-0924-9_11

Download citation

  • DOI: https://doi.org/10.1007/978-94-010-0924-9_11

  • Publisher Name: Springer, Dordrecht

  • Print ISBN: 978-0-7923-6696-6

  • Online ISBN: 978-94-010-0924-9

  • eBook Packages: Springer Book Archive

Publish with us

Policies and ethics

Search

Navigation