title: Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase.
abstract: We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase-inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was approximately 10-fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild-type, and ninefold higher activity towards soman. L69V exhibited 100-fold faster hydrolysis of DFP than the wild-type. The active-site mutant H115W exhibited 270-380-fold enhancement toward hydrolysis of the P-S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino-acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP-pNP, indicating that PON1 is less active toward the more toxic optical isomers.
authors.affiliation: Division of Medicinal Chemistry, Israel Institute for Biological Research, Ness Ziona, Israel. amitai@iibr.gov.il
authors.forename: Gabriel
authors.initials: G
authors.lastname: Amitai
authors.forename: Leonid
authors.initials: L
authors.lastname: Gaidukov
authors.forename: Rellie
authors.initials: R
authors.lastname: Adani
authors.forename: Shelly
authors.initials: S
authors.lastname: Yishay
authors.forename: Guy
authors.initials: G
authors.lastname: Yacov
authors.forename: Moshe
authors.initials: M
authors.lastname: Kushnir
authors.forename: Shai
authors.initials: S
authors.lastname: Teitlboim
authors.forename: Michal
authors.initials: M
authors.lastname: Lindenbaum
authors.forename: Peter
authors.initials: P
authors.lastname: Bel
authors.forename: Olga
authors.initials: O
authors.lastname: Khersonsky
authors.forename: Dan S
authors.initials: DS
authors.lastname: Tawfik
authors.forename: Haim
authors.initials: H
authors.lastname: Meshulam
mesh_terms: D000818:Animals; D043303:Aryldialkylphosphatase / Q000097:blood* / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology; D049832:Decapodiformes / Q000201:enzymology; D019020:Directed Molecular Evolution; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000502:physiology; D044345:Phosphoric Triester Hydrolases / Q000502:physiology; D011549:Pseudomonas / Q000201:enzymology; D012999:Soman / Q000378:metabolism / Q000633:toxicity; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009943:Organophosphorus Compounds; D012999:Soman; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases; D044345:Phosphoric Triester Hydrolases; D043303:Aryldialkylphosphatase; C020061:diisopropyl-fluorophosphatase; C053914:cyclohexyl methylphosphonofluoridate