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 $ 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 wildtype. 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.

In the light of directed evolution: Pathways of adaptive protein evolution

“…A particularly compelling example comes from phosphotriesterase and lactonase enzymes. These enzymes have been engineered for efficient activity on several new substrates through selection of mutations that enhance existing promiscuous functions (26,40,41). Complementary work has shown that selection for a promiscuous activity likely explains the natural origin of a bacterial enzyme that hydrolyzes a synthetic compound only recently introduced into the environment (42).…”

Paraoxonase status and plasma butyrylcholinesterase activity in chlorpyrifos manufacturing workers

“…The potential implication of the PON1 192 alloforms is complicated, because some OP substrates are hydrolyzed with greater catalytic efficiency by the PON1 R192 alloform than by the PON1 Q192 alloform, whereas other substrates show an opposite activity (Billecke et al, 2000;Amitai et al, 2006). For example, the PON1 192R alloform hydrolyzes paraoxon in vitro with greater catalytic efficiency than does the PON1 192Q alloform, whereas the PON1 192 alloforms hydrolyze diazinon oxon (diazoxon) with nearly equivalent catalytic efficiencies (Haley et al, 1999;Furlong et al, 2000b;Lee et al, 2003).…”

Degradation of nerve agents by an organophosphate-degrading agent (OpdA)

“…In the present work, the concentration of residual nerve agent was determined, and an adaptation of the methods of Hammond and Forster [15] and Amitai et al [16] was chosen. This method is based on the extent of inhibition of acetylcholinesterase activity in a given time period, as measured using the Ellman assay [12].…”