tailieunhanh - Báo cáo khoa học: Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase

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 diminutaOP hydrolase (OPH) and squidO,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). | iFEBS Journal Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase Gabriel Amitai1 Leonid Gaidukov2 Rellie Adani1 Shelly Yishay1 Guy Yacov1 Moshe Kushnir1 Shai Teitlboim1 Michal Lindenbaum1 Peter Bel1 Olga Khersonsky2 Dan S. Tawfik2 and Haim Meshulam1 1 Division of MedicinalChemistry Israel Institute for BiologicalResearch Ness Ziona Israel 2 Department of BiologicalChemistry Weizmann Institute of Science Rehovot Israel Keywords acetylcholinesterase detoxification organophosphates paraoxanase stereoselective degradation Correspondence G. Amitai Department of Pharmacology IIBR PO Box 19 Ness Ziona 74100 Israel Fax 972 8 938 1559 Tel 972 8 938 1591 E-mail amitai@ Received 4 September 2005 revised 16 February 2006 accepted 23 February 2006 doi 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 ơ ơ-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 10-fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo respectively compared with .