Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad

Bella L. Grigorenko, Dana A. Novichkova, Sofya V. Lushchekina, Irina V. Zueva, Lawrence M. Schopfer, Alexander V. Nemukhin, Sergey D. Varfolomeev, Oksana Lockridge, Patrick Masson

Research output: Contribution to journalArticle

Abstract

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

Original languageEnglish (US)
Pages (from-to)138-146
Number of pages9
JournalChemico-Biological Interactions
Volume306
DOIs
StatePublished - Jun 1 2019

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Butyrylcholinesterase
Organophosphates
Enzymes
Electrophoresis
Cell culture
Culture Media
Cell Culture Techniques
Staining and Labeling
Native Polyacrylamide Gel Electrophoresis
Phosphorylation
Molecular Dynamics Simulation
Computer Simulation
Molecular dynamics
Polyacrylamide Gel Electrophoresis
Substrates

Keywords

  • Butyrylcholinesterase
  • Catalytic triad
  • Computer-designed enzyme
  • Markov state model
  • Molecular dynamics
  • Organophosphate inhibition/reactivation
  • QM/MM

ASJC Scopus subject areas

  • Toxicology

Cite this

Grigorenko, B. L., Novichkova, D. A., Lushchekina, S. V., Zueva, I. V., Schopfer, L. M., Nemukhin, A. V., ... Masson, P. (2019). Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad. Chemico-Biological Interactions, 306, 138-146. https://doi.org/10.1016/j.cbi.2019.04.019

Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad. / Grigorenko, Bella L.; Novichkova, Dana A.; Lushchekina, Sofya V.; Zueva, Irina V.; Schopfer, Lawrence M.; Nemukhin, Alexander V.; Varfolomeev, Sergey D.; Lockridge, Oksana; Masson, Patrick.

In: Chemico-Biological Interactions, Vol. 306, 01.06.2019, p. 138-146.

Research output: Contribution to journalArticle

Grigorenko, BL, Novichkova, DA, Lushchekina, SV, Zueva, IV, Schopfer, LM, Nemukhin, AV, Varfolomeev, SD, Lockridge, O & Masson, P 2019, 'Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad', Chemico-Biological Interactions, vol. 306, pp. 138-146. https://doi.org/10.1016/j.cbi.2019.04.019
Grigorenko, Bella L. ; Novichkova, Dana A. ; Lushchekina, Sofya V. ; Zueva, Irina V. ; Schopfer, Lawrence M. ; Nemukhin, Alexander V. ; Varfolomeev, Sergey D. ; Lockridge, Oksana ; Masson, Patrick. / Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad. In: Chemico-Biological Interactions. 2019 ; Vol. 306. pp. 138-146.
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AU - Grigorenko, Bella L.

AU - Novichkova, Dana A.

AU - Lushchekina, Sofya V.

AU - Zueva, Irina V.

AU - Schopfer, Lawrence M.

AU - Nemukhin, Alexander V.

AU - Varfolomeev, Sergey D.

AU - Lockridge, Oksana

AU - Masson, Patrick

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N2 - A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

AB - A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

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