Directed evolution of hydrolases for prevention of G-type nerve agent intoxication

Area of Science:

• Biochemistry
• Enzyme Engineering
• Toxicology

Background:

• Organophosphate nerve agents are highly toxic, necessitating rapid detoxification methods.
• Serum paraoxonase (PON1) is a potential bioscavenger but exhibits low catalytic efficiency against toxic G-agent isomers.
• Effective bioscavenging requires enzymes with catalytic efficiencies exceeding 10^7 M(-1) min(-1).

Purpose of the Study:

• To enhance the catalytic efficiency of serum paraoxonase (PON1) for organophosphate nerve agent detoxification.
• To develop novel high-throughput screening methods for identifying improved PON1 variants.
• To demonstrate the in vivo efficacy of engineered PON1 for nerve agent prophylaxis.

Main Methods:

• Combined random and targeted mutagenesis of PON1.
• High-throughput screening using fluorogenic analogs in emulsion compartments.
• Direct screening for acetylcholinesterase protection against nerve agent inactivation.
• In vivo testing of evolved PON1 variants for prophylactic activity.

Main Results:

• Achieved a ~10^5-fold enhancement in PON1 activity toward a cyclosarin analog.
• Isolated PON1 variants with k(cat)/K(M) ~ 10^7 M(-1) min(-1) against toxic G-agent isomers and cyclosarin.
• Demonstrated successful in vivo prophylactic activity of an engineered PON1 variant.

Conclusions:

• Engineered PON1 variants exhibit significantly improved catalytic efficiency for organophosphate nerve agent detoxification.
• Novel screening platforms facilitate the rapid evolution of highly effective bioscavenging enzymes.
• These advancements provide a foundation for developing PON1-based prophylaxis against G-type nerve agents.