Mutagenesis of organophosphorus hydrolase to enhance hydrolysis of the nerve agent VX.
Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.
Gopal
S
S
Soil Microbial Systems Laboratory, ARS/USDA, Beltsville, Maryland, 20705, USA.
Rastogi
V
V
Ashman
W
W
Mulbry
W
W
D000110:Acetylcholinesterase / Q000737:chemistry; D019943:Amino Acid Substitution; D043303:Aryldialkylphosphatase; D001483:Base Sequence; D001673:Biodegradation, Environmental; D002619:Chemical Warfare Agents / Q000493:pharmacokinetics*; D017931:DNA Primers; D004950:Esterases / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D007306:Insecticides / Q000493:pharmacokinetics*; D007531:Isoflurophate / Q000493:pharmacokinetics; D016297:Mutagenesis, Site-Directed; D063086:Organothiophosphates / Q000493:pharmacokinetics; D009946:Organothiophosphorus Compounds / Q000493:pharmacokinetics*; D010261:Paraoxon / Q000493:pharmacokinetics; D010278:Parathion / Q000493:pharmacokinetics; D004849:Phenylphosphonothioic Acid, 2-Ethyl 2-(4-Nitrophenyl) Ester / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D000110:Acetylcholinesterase; D043303:Aryldialkylphosphatase; D002619:Chemical Warfare Agents; D017931:DNA Primers; D004950:Esterases; D007306:Insecticides; D007531:Isoflurophate; D063086:Organothiophosphates; D009946:Organothiophosphorus Compounds; D010261:Paraoxon; D010278:Parathion; D004849:Phenylphosphonothioic Acid, 2-Ethyl 2-(4-Nitrophenyl) Ester; D011994:Recombinant Proteins; C009680:VX-agent; C030064:demeton-S-methyl