title: Directed evolution of hydrolases for prevention of G-type nerve agent intoxication.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Rinkoo D
authors.initials: RD
authors.lastname: Gupta
authors.forename: Moshe
authors.initials: M
authors.lastname: Goldsmith
authors.forename: Yacov
authors.initials: Y
authors.lastname: Ashani
authors.forename: Yair
authors.initials: Y
authors.lastname: Simo
authors.forename: Gavriel
authors.initials: G
authors.lastname: Mullokandov
authors.forename: Hagit
authors.initials: H
authors.lastname: Bar
authors.forename: Moshe
authors.initials: M
authors.lastname: Ben-David
authors.forename: Haim
authors.initials: H
authors.lastname: Leader
authors.forename: Raanan
authors.initials: R
authors.lastname: Margalit
authors.forename: Israel
authors.initials: I
authors.lastname: Silman
authors.forename: Joel L
authors.initials: JL
authors.lastname: Sussman
authors.forename: Dan S
authors.initials: DS
authors.lastname: Tawfik
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism; D043303:Aryldialkylphosphatase / Q000378:metabolism; D055162:Biocatalysis; D002619:Chemical Warfare Agents / Q000506:poisoning*; D019020:Directed Molecular Evolution; D004655:Emulsions; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D011041:Poisoning / Q000517:prevention & control*
chemical_list: D002619:Chemical Warfare Agents; D004655:Emulsions; D006867:Hydrolases; D000110:Acetylcholinesterase; D043303:Aryldialkylphosphatase