Enzymatic Neutralization of the Chemical Warfare Agent VX: Evolution of Phosphotriesterase for Phosphorothiolate Hydrolysis

Abstract

The V-type nerve agents (VX and VR) are among the most toxic substances known. The high toxicity and environmental persistence of VX makes the development of novel decontamination methods particularly important. The enzyme phosphotriesterase (PTE) is capable of hydrolyzing VX but with an enzymatic efficiency more than 5-orders of magnitude lower than with its best substrate, paraoxon. PTE has previously proven amenable to directed evolution for the improvement of catalytic activity against selected compounds through the manipulation of active site residues. Here, a series of sequential two-site mutational libraries encompassing twelve active site residues of PTE was created. The libraries were screened for catalytic activity against a new VX analogue (DEVX), which contains the same thiolate leaving group of VX coupled to a di-ethoxy phosphate core rather than the ethoxy, methylphosphonate core of VX. The evolved catalytic activity with DEVX was enhanced 26-fold relative to wildtype PTE. Further improvements were facilitated by targeted error-prone PCR mutagenesis of Loop-7 and additional PTE variants were identified with up to a 78-fold increase in the rate of DEVX hydrolysis. The best mutant hydrolyzed the racemic nerve agent VX with a value of kcat/Km of 7×104 M−1 s−1; a 230-fold improvement relative to the wild-type PTE. The highest turnover number achieved by the mutants created for this investigation was 137 s−1; an enhancement of 152-fold relative to wild-type PTE. The stereoselectivity for the hydrolysis of the two enantiomers of VX was relatively low. These engineered mutants of PTE are the best catalysts ever reported for the hydrolysis of nerve agent VX.

Variants of Phosphotriesterase for the Enhanced Detoxification of the Chemical Warfare Agent VR

“…[7][8][9][10][11][12] Among these enzymes only PTE and PON1 are known to hydrolyze the V-type nerve agents. Wild-type PTE was used as the main ingredient in DEFENZ, which was 5 marketed by Genencor for the decontamination of organophosphate nerve agents.…”

“…7 Mutation of residues contained within the active site of PTE resulted in the isolation of the variant H245Q/H257F (QF), which exhibited a 100-fold improvement for the hydrolysis of VX, 6 relative to the wild-type enzyme (see Table 1 for identity of variants). 7 Additional active site variations led to the identification of the mutant CVQFL (QF + I106C/F132V/S308L) with a similar catalytic efficiency for the hydrolysis of VX, but a three-fold improvement in kcat. The best variant identified to date for the hydrolysis of VX is VRN-VQFL (QF + F132V/S308L + A80V/K185R/I274N).…”

Recent advances in enzyme promiscuity

“…For further improvement, loop7 of Mutant PTE was mutated by error-prone PCR, which resulted in up to 78-fold increase in the rate of DEVX hydrolysis and 230-fold improvement in hydrolyzing racemic nerve agent VX as compare to wild-type wildtype PTE. However, stereo-selectivity for the hydrolysis of the two enantiomers of VX was relatively low [44]. The catalytic activity of PTE from A. radiobacter, for the common organophosphorous insecticide malathion was enhanced by changing serine to leucine at position 308 and tyrosine to alanine at position 309, which resulted in 5000-fold increase in Kcat/Km value.…”

“…Promiscuous bacterial phosphotriesterases (PTEs) are capable of hydrolyzing VX but with very low level of activity [44]. PTE mutant library was created by mutating 12 active-site residues of PTE in order to enhance its catalytic efficiency.…”

Directed Protein Packaging within Outer Membrane Vesicles from <em>Escherichia coli</em>: Design, Production and Purification

“…Prolonged or significant exposure to organophosphates commonly results in uncontrollable convulsions and typically causes death via asphyxiation. While PTE exhibits the highest catalytic activity toward paraoxon, a very potent insecticide, it is also capable of hydrolyzing a broad range of other pesticides and V/G type chemical nerve agents 16 . To facilitate OMV packaging, a bacterial plasmid was designed that encodes a gene construct that contains an inducible promoter, a periplasmic localization sequence, and a short multiple cloning site upstream of the SC gene sequence.…”