Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although ...serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by ∼10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ∼ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
One of the major challenges in dealing with chemical warfare agent (CWA) dispersal, whether in the battlefield or after a terror act, is decontamination and rehabilitation of any contaminated area. ...Organophosphates (OPs) are considered to be among the deadliest CWAs to date. Other OPs are used as pesticides in modern agriculture, and are considered environmentally hazardous. Current methods for OP decontamination are either dangerous or insufficiently effective. As a promising solution for this problem, bioremediation-the use of biocomponents for environmental remediation-is a potentially effective, safe, and environment-friendly method. The technology relies on several enzymatic mechanisms, and can be applied in various ways. We will review recent achievements and potential applications, such as biocatalyst-containing foams and an enzymatic sponge, for environmental as well as personal exterior decontamination.
A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously ...enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic SP isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.2-5 × 107 M−1 min−1. Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 × 10−4 in favor of the RP isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the SP isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.
► Directed evolution enhanced PON1's rates of G-agent hydrolysis up to 340-fold ► Enzyme variants that effectively hydrolyze all major G-type nerve agents ► Catalytic efficiency enough for prophylactic protection against the major G-agents ► A complete reversion of the stereospecificity
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