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

Area of Science:

• Biochemistry
• Enzyme Engineering
• Computational Chemistry

Background:

• Human butyrylcholinesterase (BChE) is crucial for hydrolyzing organophosphates.
• Existing BChE mutants have limitations in catalytic efficiency and stability.
• Novel enzyme designs are needed for improved organophosphate detoxification.

Purpose of the Study:

• To design and characterize a computer-generated mutant of human BChE with a modified catalytic triad.
• To assess the mutant's reactivity towards organophosphates using computational and experimental methods.
• To establish a template for creating novel BChE variants with enhanced hydrolase activity.

Main Methods:

• In silico design of a double mutant (N322E/E325G) with a novel catalytic triad (S198·H438·N322E).
• Molecular dynamics and Markov state model analysis to predict triad operability.
• Quantum mechanics/molecular mechanics (QM/MM) modeling for reactivity assessment.
• Expression of a truncated monomeric mutant in Expi293 cells and activity staining.

Main Results:

• Computational analyses predicted the functionality of the novel catalytic triad in the N322E/E325G mutant.
• QM/MM modeling indicated high reactivity of the mutant towards echothiophate.
• Activity staining confirmed the enzymatic activity of the expressed monomeric BChE mutant.
• The mutant demonstrated slow self-reactivation after echothiophate inhibition.

Conclusions:

• The computer-designed N322E/E325G BChE mutant with a new catalytic triad is functional.
• This mutant serves as a promising template for developing novel human BChE variants with organophosphate hydrolase activity.
• Further studies are needed to determine precise kinetic parameters due to cellular background interference.