Applied Microbiology and Biotechnology

, Volume 100, Issue 13, pp 5829–5838 | Cite as

Extensive hydrolysis of phosphonates as unexpected behaviour of the known His6-organophosphorus hydrolase

  • Ilya V. Lyagin
  • Mariia S. Andrianova
  • Elena N. Efremenko
Biotechnologically relevant enzymes and proteins


The catalytic activity of hexahistidine-tagged organophosphorus hydrolase (His6-OPH) in hydrolytic reactions of methylphosphonic acid (MPA) and its monoesters and diesters being decomposition products of R-VX was demonstrated for the first time. The catalytic constants of enzyme in such reactions were determined. The mechanism of C–P bond cleavage in the MPA by His6-OPH was proposed. Such reaction was estimated to be carried out with the soluble and nanocapsulated forms of His6-OPH. His6-OPH was demonstrated to be capable of degrading the key organophosphorus components of reaction masses (RMs) that are produced by the chemical detoxification of R-VX and RMs are multi-substrate mixtures for this enzyme. The kinetic model describing the behaviour of His6-OPH in RMs was proposed and was shown to adequately fit experimental points during degradation of the real samples of RMs.


Organophosphorus compound Phosphonate Mechanism Enzymatic degradation Hexahistidine-tagged organophosphorus hydrolase 



This work was financially supported by the Ministry of Education and Science of RF (contract no. 02.515.11.5002). Enzyme-polyelectrolyte complexes were obtained with financial support by the Russian Foundation for Basic Research (grant no. 15-54-54011). Authors extend special thanks to research fellows of 27 Research Center of MD RF (Moscow, Russia) and State Research Institute of Organic Chemistry and Technology (Moscow, Russia) for supporting the work as analytic maintenance and supplying with substrates. Also, authors are grateful to Viktor I. Kholstov (Department of Realization of Conventional Obligation, Ministry of Industry and Trade of RF, Moscow, Russia) for supporting the work in toto.

Compliance with ethical standards


This work was financially supported by the Ministry of Education and Science of RF (contract no. 02.515.11.5002) and by the Russian Foundation for Basic Research (grant no. 15–54-54,011).

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7407_MOESM1_ESM.pdf (200 kb)
ESM 1 (PDF 199 kb)


  1. Ashkenazi N, Segall Y, Chen R, Sod-Moriah G, Fattal E (2010) The mechanism of nucleophilic displacements at phosphorus in chloro-substituted methylphosphonate esters: P-O vs P-C bond cleavage: a DFT study. J Org Chem 75:1917–1926. doi: 10.1021/jo9026325 CrossRefPubMedGoogle Scholar
  2. Centers for Disease Control and Prevention, Department of Health and Human Services Review of the U.S. (2005) Army proposal for off-site treatment and disposal of caustic Vx hydrolysate from the Newport Chemical Agent Disposal Facility. A report to Congress, Washington DCGoogle Scholar
  3. Christol H, Marty C (1968) Hydrolyse basique de phosphonates. II Étude Quantitative. J Organomet Chem 12:471–478. doi: 10.1016/S0022-328X(00)88700-8 CrossRefGoogle Scholar
  4. Efremenko EN, Votchitseva YA, Aliev TK, Varfolomeev SD (2005) Recombinant plasmid DNA ptes-his-oph and producer of oligohistidine-containing organophosphate hydrolase. Russian Patent 2255975Google Scholar
  5. Efremenko E, Votchitseva Y, Plieva F, Galaev I, Mattiasson B (2006) Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography. Appl Microbiol Biot 70:558–563. doi: 10.1007/s00253-005-0103-x CrossRefGoogle Scholar
  6. Efremenko EN, Votchitseva JA, Kurochkin IN, Varfolomeev SD, Gachok IV, Zav’jalova NV, Kapashin VP, Kholstov VI (2007) Method of the zymohydrolysis of the organophosphorous combat poisonous substances. Russian Patent 2296164Google Scholar
  7. Ember LR (2006) Chemical weapons deadline at risk. Chem Eng News 84:27–30. doi: 10.1021/cen-v084n016.p027 Google Scholar
  8. Freedman LD, Doak GO (1957) The preparation and properties of phosphonic acids. Chem Rev 57:479–523. doi: 10.1021/cr50015a003 CrossRefGoogle Scholar
  9. Ghanem E, Li Y, Xu C, Raushel FM (2007) Characterization of a phosphodiesterase capable of hydrolyzing EA 2192, the most toxic degradation product of the nerve agent VX. Biochemistry 46:9032–9040. doi: 10.1021/bi700561k CrossRefPubMedGoogle Scholar
  10. Gudkov DA, Lyagin IV, Efremenko EN, Kabanov AV (2012) Effect of dimerization on the catalytic properties of native and chimeric organophosphorus hydrolase determined by molecular modeling of the enzyme structure. Russ Chem Bull 61:449–455. doi: 10.1007/s11172-012-0062-1 CrossRefGoogle Scholar
  11. Gupta RC (ed) (2009) Handbook of toxicology of chemical warfare agents. Academic Press, LondonGoogle Scholar
  12. Hilderbrand RL (1983) The role of phosphonates in living system. CRC Press, Boca Raton, FLGoogle Scholar
  13. Horiguchi M, Kandatsu M (1959) Isolation of 2-aminoethane phosphonic acid from rumen protozoa. Nature 184:901–902. doi: 10.1038/184901b0 CrossRefPubMedGoogle Scholar
  14. Kabanov AV, Efremenko EN, Kljachko NL, Bronich TK, Lyagin IV, Varfolomeev SD (2014) Nanoscale enzyme biocatalyst for in vivo detoxification of organophosphorous compounds. Russian Patent 2525658Google Scholar
  15. Li WS, Lum KT, Chen-Goodspeed M, Sogorb MA, Raushel FM (2001) Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase. Bioorg Med Chem 9:2083–2091. doi: 10.1016/S0968-0896(01)00113-4 CrossRefPubMedGoogle Scholar
  16. Lyagin IV, Efremenko EN, Kabanov AV (2014) Catalytic characteristics of enzyme-polyelectrolyte complexes based on hexahistidine-containing organophosphorus hydrolase. Mosc Univ Chem Bull 69:125–130. doi: 10.3103/S0027131414030055 CrossRefGoogle Scholar
  17. McGrath JW, Chin JP, Quinn JP (2013) Organophosphonates revealed: new insights into the microbial metabolism of ancient molecules. Nat Rev Microbiol 11:412–419. doi: 10.1038/nrmicro3011 CrossRefPubMedGoogle Scholar
  18. Metcalf WW, Griffin BM, Cicchillo RM, Gao J, Janga SC, Cooke HA, Circello BT, Evans BS, Martens-Habbena W, Stahl DA, van der Donk WA (2012) Synthesis of methylphosphonic acid by marine microbes: a source for methane in the aerobic ocean. Science 337:1104–1107. doi: 10.1126/science.1219875 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mikolajczyk M, Midura W, Grzejszczak S (1984) Synthesis of mono- and 1,4-dicarbonyl compounds based on the oxygenation of phosphonate carbanions. Synthesis of dihydrojasmone, allethrone and methylenomycin b. Tetrahedron Lett 25:2489–2492. doi: 10.1016/S0040-4039(01)81212-0 CrossRefGoogle Scholar
  20. Munro NB, Talmage SS, Griffin GD, Waters LC, Watson AP, King JF, Hauschild V (1999) The sources, fate, and toxicity of chemical warfare agent degradation products. Environ Health Persp 107:933–974. doi: 10.1289/ehp.99107933 CrossRefGoogle Scholar
  21. Nash T (1953) The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem J 55:416–421. doi: 10.1042/bj0550416 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Organisation for the Prohibition of Chemical Weapons (2005) Convention on the prohibition of the development, production, stockpiling and use of chemical weapons and on their destruction. Hague, NetherlandsGoogle Scholar
  23. Organisation for the Prohibition of Chemical Weapons (2014) Report of the OPCW on the Implementation of the Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction in 2013. Conference of the States Parties, Nineteenth Session, 1–5 December 2014, Hague, Netherlands, CS-2014-8970(E)Google Scholar
  24. Peel JL, Loughman BC (1957) Some observations on the role of copper ions in the reduction of phosphomolybdate by ascorbic acid and their application in the determination of inorganic orthophosphate. Biochem J 65:709–716. doi: 10.1042/bj0650709 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Rabinowitz R (1968) Synthesis of monoesters of phosphonic acids. J Am Chem Soc 82:4564–4567. doi: 10.1021/ja01502a030 CrossRefGoogle Scholar
  26. Razumovsky SD, Efremenko EN, Makhlis TA, Senko OV, Ya Bikhovsky M, Podmaster’ev VV, Varfolomeev SD (2008) Effect of immobilization on the main dynamic characteristics of the enzymatic oxidation of methane to methanol by bacteria Methylosinus sporium B-2121. Russ Chem Bull 57:1633–1636. doi: 10.1007/s11172-008-0211-8 CrossRefGoogle Scholar
  27. Sergeeva V, Efremenko E, Kazankov G, Varfolomeyev S (2000) Double effect of organic amines (activation and inhibition) on the phosphotriesterase. J Mol Catal B Enzym 10:571–576. doi: 10.1016/S1381-1177(00)00102-8 CrossRefGoogle Scholar
  28. Small MJ (1984) Compounds formed from the chemical decontamination of HD, GB, and VX and their environmental fate. Tech rpt 8304, DTIC accession no. AD-A149515, U.S. Army Medical Bioengineering research and Development Laboratory, Fort Detrick, MDGoogle Scholar
  29. Sunden H, Engqvist M, Casas J, Ibrahem I, Cordova A (2004) Direct aminoacid catalyzed asymmetric α-oxidation of ketons with molecular oxygen. Angew Chem Int Ed Engl 43:6532–6535. doi: 10.1002/anie.200460295 CrossRefPubMedGoogle Scholar
  30. Utkin AJ, Pyzh’janov IV, Sheluchenko VV, Petrunin VA, Kapashin VP, Kholstov VI, Kondrat’ev VB (2009) Method of destruction of chemical ammunition, equipped with phosphoroorganic chemical agents and having technological threaded holes in case. Russian Patent 2352375Google Scholar
  31. Votchitseva YA, Efremenko EN, Aliev TK, Varfolomeyev SD (2006) Properties of hexahistidine-tagged organophosphate hydrolase. Biochemistry (Mosc) 71:167–172. doi: 10.1134/S0006297906020088 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Chemistry DepartmentLomonosov Moscow State UniversityMoscowRussia
  2. 2.Institute of Biochemical Physics RASMoscowRussia
  3. 3.SMC Technological Center MIETZelenogradRussia

Personalised recommendations