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Archives of Toxicology

, Volume 90, Issue 11, pp 2711–2724 | Cite as

Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro

  • Moshe Goldsmith
  • Simone Eckstein
  • Yacov Ashani
  • Per GreisenJr.
  • Haim Leader
  • Joel L. Sussman
  • Nidhi Aggarwal
  • Sergey Ovchinnikov
  • Dan S. Tawfik
  • David Baker
  • Horst Thiermann
  • Franz Worek
Molecular Toxicology

Abstract

The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23’s ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence–activity relationships along the pathway of PTE’s laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M−1 min−1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence–activity relationships.

Keywords

Organophosphorus compounds Organophosphate hydrolase Phosphotriesterase Bioscavenger Mutant Detoxification 

Notes

Acknowledgments

The study was funded by the German Ministry of Defence. The authors wish to thank Dr. Shira Albeck from the Israel Structural Proteomics Center (ISPC), Rehovot, Israel, for the expression and purification of the PTE variants and are grateful to M. Baumann and T. Hannig for their expert technical assistance. PJG was supported by Carlsberg-fondet and EMBO Long-term postdoctoral fellowship. PTE variants were engineered and studied under a DTRA Project Grant (HDTRA1-11-C-0026),

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

204_2015_1626_MOESM1_ESM.docx (4.6 mb)
Supplementary material 1 (DOCX 4760 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Moshe Goldsmith
    • 2
  • Simone Eckstein
    • 1
  • Yacov Ashani
    • 2
  • Per GreisenJr.
    • 3
  • Haim Leader
    • 4
  • Joel L. Sussman
    • 5
  • Nidhi Aggarwal
    • 5
  • Sergey Ovchinnikov
    • 3
  • Dan S. Tawfik
    • 2
  • David Baker
    • 3
  • Horst Thiermann
    • 1
  • Franz Worek
    • 1
  1. 1.Bundeswehr Institute of Pharmacology and ToxicologyMunichGermany
  2. 2.Department of Biological ChemistryWeizmann Institute of ScienceRehovotIsrael
  3. 3.Institute for Protein Design and Department of BiochemistryUniversity of WashingtonSeattleUSA
  4. 4.Department of Materials and InterfacesWeizmann Institute of ScienceRehovotIsrael
  5. 5.Department of Structural BiologyWeizmann Institute of ScienceRehovotIsrael

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