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

Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase Gabriel Amitai 1 , Leonid Gaidukov 2 , Rellie Adani 1 , Shelly Yishay 1 , Guy Yacov 1 , Moshe Kushnir 1 , Shai Teitlboim 1 , Michal Lindenbaum 1 , Peter Bel 1 , Olga Khersonsky 2 , Dan S. Tawfik 2 and Haim Meshulam 1 1 Division of Medicinal Chemistry, Israel Institute for Biological Research, Ness Ziona, Israel 2 Department of Biological Chemistry, 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@iibr.gov.il (Received 4 September 2005, revised 16 February 2006, accepted 23 February 2006) doi:10.1111/j.1742-4658.2006.05198.x 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 non- hydrolyzed OP. As stereoisomers of cyclosarin and soman differ signifi- cantly 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 wild- type. The active-site mutant H115W exhibited 270–380-fold enhancement toward hydrolysis of the P–S bond in parathiol, a phosphorothiolate ana- log 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. Abbreviations ChPo, chlorpyrifos-oxon [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl)phosphate]; cyclosarin, O-cyclohexyl methylphosphonofluoridate; DEPCyC: O,O-diethyl phosphate O-(3-cyano-7-coumarinyl); DFP, O,O-di-isopropyl fluorophosphate; IMP-pNP, O-isopropyl O-(p-nitrophenyl)methyl- phosphonate; OPAA, organophosphorus acid anhydrolase; OP, organophosphate; OPH, organophosphate hydrolase; paraoxon, O,O-diethyl O-(p-nitrophenyl) phosphate; parathiol, O,O-diethyl S-(p-nitrophenyl) phosphorothiolate; PC, the annotation of PON1 variants screened by the phospho-coumarin DEP-CyC; PFPD, pulsed flame photometer detector; PON1, mammalian paraoxonase (EC 3.1.8.1); soman, O-pinacolyl methylphosphonofluoridate; VX, O-ethyl S-(N,N-di-isopropylaminoethyl) methylphosphonothiolate. 1906 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research Toxic organophosphates (OPs) that serve as nerve agents, such as O,O-di-isopropyl fluorophosphate (DFP), soman and cyclosarin (O-cyclohexyl methyl- phosphonofluoridate), and various insecticides, such as chlorpyrifos, parathion and their oxo-metabolites, chlorpyrifos-oxon (ChPo) and paraoxon [O,O-diethyl O-(p-nitrophenyl) phosphate] (Scheme 1), exert their toxicity by irreversible inhibition of acetylcholinesterase [1]. Inhibition of acetylcholinesterase results in severe cholinergic toxic signs caused by increased concentra- tions of acetylcholine at cholinergic nerve–nerve and nerve–muscle synapses [1]. The treatment of OP poison- ing is based mainly on therapeutic combination of anti-cholinergic drugs such as atropine together with quaternary oxime reactivators of inhibited acetylcholin- esterase such as 2-pyridinealdoximemethiodide and tox- ogonin [2–4]. The potential use of acetylcholinesterase and butyrylcholinesterase for stoichiometric scavenging of toxic OPs and various OP hydrolases (OPHs) as cata- lytic scavengers has been studied extensively [5–8]. OPHs could also be used for noncorrosive decontam- ination of sensitive surfaces including human skin [9]. Four groups of hydrolases have been studied with regard to OP degradation: (a) bacterial (Pseudomonas diminuta or Flavobacterium sp.) OPH (also known as phosphotriesterase) was cloned and exhibited hydrolytic activity toward various nerve agents [10]; (b) organo- phosphorus acid anhydrolase (OPAA) from Alteromon- as sp. JD6.5 [12], a halophilic prolidase that exhibits marked hydrolytic activity toward soman, DFP and cyclosarin [13]; (c) recombinant Loligo vulgaris squid DFPase cloned by Scharff et al. [14] is active toward DFP and other toxic OP compounds; (d) mammalian serum paraoxonases (PON1), isolated from human, and other mammalian sera. PON1 is a group of calcium- dependent hydrolases capable of catalyzing the hydro- lysis of various lactones, esters and certain OP compounds [11]. The human serum paraoxonase ⁄ arylesterase gene (PON1) is a member of a multigene family [15], the primary function of which appears to be lactonase [27–29]. The hydrolysis of OPs, including paraoxon which gave PON1 its name, turned out to be a promiscuous activity of PON1 [20,27,29]. The rate of hydrolysis of certain nerve agents such as sarin and soman by human serum PON1 is comparable to that of Ps. diminuta OPH, with bimolecular rate constants (k cat ⁄ K m )of10 5 )10 6 m )1 Æmin )1 [16]. The catalytic effi- ciency of PON1 in the hydrolysis of sarin and soman and the possibility to re-inject it in humans render PON1 a possible candidate for medical countermeasure against nerve agent poisoning [16]. Pertinently, it was estimated that a 10-fold increase in wild-type PON1 cat- alytic activity toward toxic OPs would be sufficient to provide substantial in vivo protection against certain nerve agents [17]. It was also noted recently that bacter- ial OPAA and OPH catalyze preferentially the hydro- lysis of the less toxic optical isomer of cyclosarin [30]. The 3D structure of mammalian PON1 was described at 2.2 A ˚ resolution [18]. It is a six-bladed b-propeller with a unique active-site lid which seems also to be involved in high-density lipoprotein binding [18]. Interestingly, the 3D structures of DFPase and PON1 are similar, Scheme 1. Chemical structure of toxic OP substrates. G. Amitai et al. Enhanced stereoselective OP hydrolysis by PON1 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research 1907 both showing a secondary structure of a six-bladed b-propeller [14,18]. Using directed evolution, various variants of PON1 were generated by Aharoni et al. [19]. The first series of PON1 variants were evolved for heterologous expression in Escherichia coli and exhibit enzymatic properties that are essentially identical with the serum-purified PON1 [19]. The recombinant vari- ants were subjected to further mutation and selection with the aim of increasing their activity towards various substrates [18–20]. In particular, a series of PON1 vari- ants were selected after three generations of enhanced evolution using the fluorogenic OP ester O,O-diethyl- phosphate O-(3-cyano-7-coumarinyl) (DEPCyC) which resembles in its structure the oxo-metabolite of the insecticide coumaphos. Certain newly evolved variants selected with DEPCyC exhibit improved rates of OPH activity toward DEPCyC and paraoxon compared with wild-type PON1 by factors of up to 155-fold and 10-fold, respectively [19,20]. As noted above, PON1 is a multifunction enzyme exhibiting lactonase, esterase and OPH activities [19]. It was noted that different muta- tions affect differently the lactonase, esterase and OPH activity of PON1 [18,20]. The amino-acid residues that affect the OPH activity are primarily Val346, Leu69, Lys192 and Ser193, but the effect of mutations on these positions has thus far only been examined with para- oxon and DEPCyC [19]. It was therefore important to examine the newly evolved PON1 variants and evaluate their detoxification activity toward nerve agents and other toxic OPs. In this report, we demonstrate mark- edly enhanced catalytic activity of certain newly evolved mammalian PON1 variants mainly toward ChPo, DFP, cyclosarin and soman. We identify the residues that affect the rate of hydrolysis of nerve agents such as cyclosarin, DFP and soman, and mutations that dra- matically enhance their degradation. We further des- cribe the PON1 variant H115W, in which the His115 that catalyzes lactone and ester hydrolysis is mutated to Trp [21]. This variant was found to display unexpectedly high activity toward parathiol [O,O -diethyl S-(p-nitro- phenyl) phosphorothiolate], a P–S bond-containing OP. The enantioselectivity of OP hydrolysis by PON1 and some of its variants is also demonstrated here with cyclosarin, soman and the sarin analog O-isopropyl O-(p-nitrophenyl)methylphosphonate (IMP-pNP). Results Detoxification of cyclosarin and soman by bacterial OPH, squid DFPase and mammalian PON1 The rate of enzymatic hydrolysis of cyclosarin, soman, DFP, ChPo, IMP-pNP, paraoxon and parathiol (Scheme 1) was determined primarily by measuring the temporal acetylcholinesterase inhibition caused by the residual nonhydrolyzed OP. This enzymatic hydrolysis of OPs measured by the acetylcholinesterase inhibition assay actually reflects detoxification of the more toxic stereoisomers of chiral OPs. Our attempts to determine K m and k cat values for cyclosarin and soman using the acetylcholinesterase inhibition assay were unsuccessful because the rate of hydrolytic detoxification did not increase with increasing substrate concentrations. Therefore, the time-course of OP detoxification was analyzed by measuring the initial rates of hydrolysis. The first-order initial rate constant (k obs ) , was calcula- ted from the slope of the linear decrease in ln(% resid- ual OP) with time. Equal concentrations of OPs as well as OPH, DFPase and PON1 variants were used in all kinetic studies. These conditions enable the compar- ison of initial rate constants obtained for OPH, DFPase or PON1 variant relative to wild-type PON1. Thus, changes in OPH activity observed for the newly evolved PON1 variants were evaluated by the ratio k obs (mutant) ⁄ k obs (wild-type). The hydrolytic activity of recombinant PON1 toward cyclosarin was sevenfold and ninefold higher than that of squid DFPase and Ps. diminuta OPH, respectively (at 0.03 mgÆmL )1 enzyme, 10 lm cyclo- sarin, k obs ¼ 25.4 · 10 )3 , 3.8 · 10 )3 and 2.7 · 10 )3 min )1 , respectively, Fig. 1A). Furthermore, PON1 was more active than DFPase and OPH in detoxifying soman, with fourfold higher rates (at 0.03 mgÆmL )1 enzyme, 10 lm soman, k obs ¼ 7.5 · 10 )3 , 1.8 · 10 )3 and 1.7 · 10 )3 min )1 , respectively, Fig. 1B). The con- centration of each enzyme was 0.03 mgÆmL )1 or 0.75 lm (when the molecular mass of OPH, DFPase and PON1 is taken as 40 kDa) and OP substrate con- centration was 10 lm. All kinetic data obtained for detoxification of cyclosarin and soman using the ace- tylcholinesterase inhibition assay were fitted to a single exponential decay function (Figs 2 and 3). Modified rates of OP detoxification by newly evolved PON1 variants The enhanced rate of detoxification of cyclosarin and soman by wild-type PON1 compared with DFPase and OPH (Fig. 1) led us to study further PON1 and its vari- ants as catalytic OP scavengers. New PON1 variants were evolved by directed evolution using the fluorogen- ic OP substrate DEPCyC [19,20]. Nine of these variants were evaluated for their hydrolysis of cyclosarin, so- man, DFP, paraoxon, parathiol, IMP-pNP and ChPo (Scheme 1). The most rapid detoxification of cyclosarin was obtained with the single-site mutants V346A Enhanced stereoselective OP hydrolysis by PON1 G. Amitai et al. 1908 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research (1.2PC) and L69V (1.1PC): k obs ¼ 270 · 10 )3 and 250 · 10 -3 min )1 , respectively, versus 25 · 10 )3 min )1 with wild-type PON1 (Fig. 2). The double mutant L69V ⁄ S193P (2.1PC) exhibited a fourfold faster detoxi- fication rate toward cyclosarin (k obs ¼ 92 · 10 )3 min )1 ; Figs 2 and 4). The variants L69V ⁄ S138L ⁄ S193P (3.1PC), L69V ⁄ S138L ⁄ S193P ⁄ N287D (3.2PC) and L69V ⁄ S138L ⁄ S193P ⁄ N287D ⁄ V346A (3.2PC ⁄ V346A) displayed 2.5–3.5-fold higher activity than wild-type PON1 (Figs 2 and 4). The rate of soman hydrolysis by wild-type PON1 was significantly slower than hydroly- sis of cyclosarin and DFP (k obs ¼ 7.5 · 10 )3 compared with 25 · 10 )3 and 17 · 10 )3 min )1 , respectively; Figs 2, 3 and 4; time-course for DFP hydrolysis is not shown). However, the variant V346A (1.2PC) exhibited a nine- fold enhancement of hydrolysis toward soman com- pared with wild-type PON1 (k obs ¼ 65 · 10 )3 and 7.5 · 10 )3 min )1 , respectively, Figs 3 and 4). In addition, the five-site mutant L69V ⁄ S138L ⁄ S193P ⁄ N287D ⁄ V346A (3.2PC ⁄ V346A) catalyzed soman detoxification twofold faster than wild-type PON1 (Figs 3 and 4). All other variants exhibited equal or slower hydrolytic rates than wild-type PON1 toward soman (Figs 3 and 4). The kinetic data obtained for en- zymatic hydrolysis of soman with all tested PON1 vari- ants indicate the importance of the V346A mutation for the enhancement of cyclosarin and soman hydro- lysis. The PON1 variant V346A also exhibited fourfold and sevenfold faster hydrolysis than wild-type PON1 toward DFP and ChPo (kinetic data not shown; see k obs ratios in Fig. 4). The most active variant toward DFP was the single-site mutant L69V, with a 100-fold enhancement over that of wild-type PON1 (k obs ¼ 1.7 versus 0.017 min )1 , Fig. 4). All other multiple mutants (with three to five active-site mutations) yielded faster rates than wild-type PON1 for DFP, cyclosarin and parathiol hydrolysis, but to a lower extent than the sin- gle and double mutants (Fig. 4). These multiple muta- tion variants also exhibited lower activity than wild-type PON1 toward soman and ChPo (Figs 3 and 4). Thus, the most universally active PON1 variant toward DFP, cyclosarin, soman and ChPo was the single-site mutant Fig. 1. Time-course of enzymatic detoxification of cyclosarin (A) and soman (B) by Ps. diminuta OPH, squid DFPase and mammalian wild- type PON1, measured by the acetylcholinesterase inhibition assay. Cyclosarin and soman concentration 10 l M;20mM Tris ⁄ HCl, pH 7.0; enzyme concentration 0.03 mgÆmL )1 (0.75 lM); CaCl 2 1mM;25°C. Initial rates of OP detoxification (k obs ,min )1 mean ± SEM, n ¼ 3) were estimated from the slopes of the linear plot of ln[% OP] versus time. All k obs values are summarized in the attached table. ND, not deter- mined. The linear plot is based on points transformed from the initial part (up to 50% of OP hydrolysis) of the experimental nonlinear curve. All kinetic experiments were performed in triplicate. The curves were fitted by one-phase exponential decay (r 2 ¼ 0.96–0.99). The plots shown are taken from one representative experiment. G. Amitai et al. Enhanced stereoselective OP hydrolysis by PON1 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research 1909 V346A, which exhibited a 4–11-fold enhanced activity compared with wild-type PON1 (Fig. 4). It was of particular interest to search for a PON1 variant that could hydrolyze parathiol, a P–S bond- containing paraoxon congener (Scheme 1) and thereby to learn about putative residues involved in the hydro- lysis of the P–S bond in OP insecticides (e.g. Demeton, malathion) and toxic nerve agents such as O-ethyl S-(N,N-di-isopropylaminoethyl) methylphosphonothio- late (VX). Therefore, the activity of PON1 variants with parathiol, paraoxon and cyclosarin was also compared at higher OP substrate concentration (100 lm) (Fig. 5). The rate of parathiol hydrolysis by wild-type PON1 was 88-fold slower than with para- oxon (k obs ¼ 6 · 10 )4 and 0.053 min )1 , respectively). However, parathiol was hydrolyzed 380-fold faster by the H115W variant (k obs ¼ 0.23 min )1 , Fig. 5) than by wild-type PON1. A complete Michaelis–Menten kinetic analysis was performed with the chromogenic symmetrical OP sub- strates paraoxon and parathiol using selected PON1 variants. Table 1 summarizes the kinetic data obtained for hydrolysis of paraoxon and parathiol by wild-type PON1 and the following variants: H115W, L69V, V346A, L69V ⁄ S138L ⁄ S193P ⁄ N287D and L69V ⁄ S138L ⁄ S193P ⁄ N287D ⁄ V346A. Figure 6 shows the kin- etics of hydrolysis of paraoxon and parathiol by H115W and wild-type PON1. It was noted that H115W enhanced the rate of parathiol hydrolysis by 270-fold compared with wild-type PON1 (k cat ⁄ K m ¼ 1.6 · 10 4 versus 60 m )1 Æs )1 ; Table 1, Fig. 7). These results corroborate those obtained for H115W with parathiol as substrate using the acetylcholinesterase inhibition assay (Figs 4 and 5). H115W enhanced paraoxon hydrolysis only 16-fold (k cat ⁄ K m ¼ 6.4 · 10 4 versus 4 · 10 3 m )1 Æs )1 ; Table 2, Fig. 7). All other PON1 variants exhibited 17–28-fold enhancement of parathiol hydrolysis compared with that of the wild- type (Table 2, Fig. 7). Similarly, these variants also showed a lower increase in activity toward paraoxon, with a 2–10-fold increase in k cat ⁄ K m values (Table 2, Fig. 7). These results strongly corroborate the data Fig. 2. Time-course of enzymatic detoxification of cyclosarin by PON1 variants measured by the acetylcholinesterase inhibition assay. Cyclo- sarin concentration 10 l M; PON1 0.03 mgÆmL )1 (0.75 lM); CaCl 2 1mM;20mM Tris ⁄ HCl, pH 7.0. All experimental kinetics data were fitted to mono-exponential decay curves drawn on the left (r 2 ¼ 0.98–0.99). Initial rate value for each PON1 variant (first-order rate constant k obs , min )1 , mean ± SEM, n ¼ 3) were calculated from the slopes of the linear plots of ln(% OP) versus time shown in the right panel. Correlation coefficients (r 2 ) for the linear plots were 0.94–0.99. The kinetic plots shown are taken from a single representative experiment out of three replicates. All k obs values are summarized in the attached table. ND, not determined. Enhanced stereoselective OP hydrolysis by PON1 G. Amitai et al. 1910 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research Fig. 3. Time-course of enzymatic degradation of Soman by PON1 variants measured by acetylcholinesterase inhibition assay. Soman concen- tration 10 l M; PON1 0.03 mgÆmL )1 (0.75 lM); CaCl 2 1mM;20mM Tris ⁄ HCl, pH 7.0. All experimental kinetics data were fitted to mono-expo- nential decay curves drawn on the left (r 2 ¼ 0.98–0.99). Initial rate values for each PON1 variant (first-order rate constant k obs ,min )1 ; mean ± SEM, n ¼ 3) were calculated from the slopes of the linear plots of ln(% OP) versus time shown on the right. Each k obs value is based on triplicate kinetic measurements. The kinetic plots shown are taken from a single representative experiment out of three replicates. All k obs values are summarized in the attached table. ND, not determined. Fig. 4. Changes in hydrolytic activity (k obs ) toward toxic OP sub- strates of PON1 variants compared with wild-type PON1 (PON1 0.03 mgÆmL )1 ,OP10lM). Detoxification was followed by residual acetylcholinesterase inhibition assay. The change in activity of each PON1 variant versus PON1 wild-type is expressed as the ratio k obs (mutant) ⁄ k obs (wild-type) drawn on a logarithmic scale. The value of this ratio for wild-type PON1 is 1. The asterisk designates a value of 1.0 obtained for the L69V variant with ChPo. Fig. 5. Changes in hydrolytic activity (k obs ) of PON1 variants using higher concentrations (100 l M) of cyclosarin, paraoxon and parathiol by PON1 variants (PON1, 0.3 mgÆmL )1 ). Detoxification was fol- lowed by acetylcholinesterase inhibition assay. The change in activ- ity is expressed as the ratio k obs (mutant) ⁄ k obs (wild-type) drawn on a logarithmic scale. G. Amitai et al. Enhanced stereoselective OP hydrolysis by PON1 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research 1911 obtained for paraoxon and parathiol using the acetyl- cholinesterase inhibition assay (Figs 4 and 5), confirm- ing that the results obtained by the acetylcholinesterase inhibition assay at single substrate concentration clearly reflect the Michaelis–Menten kinetic analysis of enzymatic activity. Stereoselective degradation of cyclosarin, soman and IMP-pNP by PON1 As indicated previously, cyclosarin is a racemic mix- ture of its S and R optical isomers configured around the phosphorus (P) atom [P(–) and P(+) optical iso- mers]. Soman is a mixture of four stereoisomers con- sisting of two pairs of diastereoisomers with two chiral centers: one on the phosphorus atom (P) and a second on the asymmetric carbon (C) atom of the pinacolyl group [P(–)C(+), P(–)C(–), P(+)C(+) and P(+)C(–) stereoisomers]. Benschop et al. [23] have noted that the pair of soman stereoisomers that are configured with the (–) isomer on the P atom [P(–)C(+ ⁄ –)] are 20–150-fold more toxic than the P(+)C(+ ⁄ –) pair of diastereoisomers. It was previ- ously noted that Ps. diminuta OPH preferentially Table 1. Michaelis–Menten analysis for the hydrolysis of paraoxon and parathiol by wild-type PON1 and its evolved variants. Each value rep- resents the mean of at least two independent experiments. Standard deviations were less then 10% of parameter values. Values in paren- theses are the x-fold increase in k cat ⁄ K m relative to the wild-type PON1. Variant Mutations Paraoxon Parathiol k cat (s )1 ) K M (mM) k cat ⁄ K M (M )1 Æs )1 ) k cat (s )1 ) K M (mM) k cat ⁄ K M (M )1 Æs )1 ) Wild-type — 6.9 1.7 4 · 10 3 0.05 0.9 60 H115W H115W 25.5 0.4 6.4 · 10 4 (16) 11 0.7 1.6 · 10 4 (270) 1.1PC L69V 11.4 0.8 1.5 · 10 4 (4) 0.8 0.7 1.1 · 10 3 (18) 1.2PC V346A 12.4 0.3 4.1 · 10 4 (10) 0.8 0.5 1.6 · 10 3 (27) 3.2PC L69V 8.0 0.2 4.0 · 10 4 (10) 0.7 0.7 1.0 · 10 3 (17) S138L S193P N287D 3.2PC ⁄ L69V 16.4 2.3 7.1 · 10 3 (2) 3.4 2.0 1.7 · 10 3 (28) V346A S138L S193P N287D V346A Fig. 6. Kinetics of hydrolysis of paraoxon (A) and parathiol (B) by the PON1 variant H115W and wild-type. Hydrolysis of OP substrates was fol- lowed by measuring the increase in p-nitrophenol A 405 at pH 8 and 25 °C. Enzymatic parameters with paraoxon and parathiol were determined by Michaelis–Menten analysis of initial rates {v 0 ¼ k cat [E] 0 [S] 0 ⁄ ([S] 0 +K M )}. Values in parentheses represent molar concentrations of PON1. Enhanced stereoselective OP hydrolysis by PON1 G. Amitai et al. 1912 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research hydrolyzes the less toxic optical isomers of cyclosarin [30] and those of p-nitrophenol analogs of sarin and soman [25]. Therefore, it was of interest to examine the stereoselectivity of cyclosarin and soman hydro- lysis exerted by PON1 variants. We compared the results of GC ⁄ pulsed flame photometer detector (PFPD) analysis, monitoring the chemical degradation of all stereoisomers of soman and cyclosarin at speci- fied time intervals, with those of the residual acetyl- cholinesterase inhibition assay, measuring its detoxification rate. Table 2 summarizes values of chemical degradation of soman and cyclosarin com- pared with its detoxification level at specified time intervals. It was noted that soman is 50% hydrolyzed by V346A within the first minute (based on GC ⁄ PFPD analysis; Table 2), whereas acetylcholinesterase inhibi- tion bioassay reveals practically no detoxification at this short time interval (1 min). Similarly, cyclosarin was degraded by 50% within the first minute (GC ⁄ PFPD analysis; Table 2) compared with less than 5% detoxification measured by acetylcholinest- erase inhibition at this short time interval (Table 2). After 100 min incubation of soman or 15 min incuba- tion of cyclosarin with V346A PON1, each agent was both degraded and detoxified by 91–98%. Soman and cyclosarin were 95–98% degraded and detoxified by wild-type PON1 only after 470 and 100 min, respect- ively. These data are consistent with faster hydrolysis of the less toxic optical isomer of cyclosarin [P(+)] and the two less toxic diastereoisomers of soman [P(+)C(+ ⁄ –)] by V346A. Stereoselective hydrolysis of chiral OP esters by PON1 was further demonstrated by using the sarin analog IMP-pNP as substrate. IMP-pNP degradation by wild-type PON1 and V346A was followed using three different analytical methods: quantitative GC ⁄ PFPD analysis, direct spec- trophotometric determination of p-nitrophenol released during hydrolysis, and detoxification kinetics measured by acetylcholinesterase inhibition assay. Table 3 summarizes the levels of degradation of Fig. 7. Changes in bimolecular rate constants (k 2 ¼ k cat ⁄ K M )of paraoxon and parathiol hydrolysis by PON1 variants compared with wild-type PON1 determined by Michaelis–Menten analysis of the enzymatic activity. The changes in activity of each variant toward degradation of paraoxon and parathiol are expressed by the ratio k 2 (mutant) ⁄ k 2 (wild-type) drawn on a logarithmic scale. Table 2. Comparison of degradation and detoxification levels of soman and cyclosarin by wild-type PON1 and V346A PON1 variant at specified time intervals. % Degradation (Deg) was determined by GC ⁄ PFPD analysis and percentage detoxification (Detox) was determined by residual acetylcholinesterase inhibition assay. Enzyme ⁄ buffer Soman Cyclosarin Time (min) % Deg % Detox Time (min) % Deg % Detox Tris, pH 7.0 1–100 < 9 < 5 1 < 9 < 5 PON1 V346A 1 50 < 5 1 50 < 5 Tris, pH 7.0 – – – 15 10 < 5 PON1 V346A 100 > 91 > 95 15 > 98 98 Tris, pH 7.0 470 30 20 100 10 10 PON1 wild-type 470 > 95 > 95 100 > 98 98 Table 3. Stereoselective hydrolysis of IMP-pNP by wild-type and V346A PON1 measured in parallel by GC ⁄ PFPD, spectrophotomet- ric and acetylcholinesterase inhibition assays. IMP-pNP concentrat- ion 10 l M; PON1 0.03 mgÆmL )1 ;50mM Tris ⁄ HCl, pH 8, 25 °C. GC analysis: samples of enzymatic degradation solutions were extract- ed at specified time intervals with equal volumes of methyl t-butyl ether that were used for quantitative GC analysis. Spectrophoto- metric analysis was performed by measuring increases in p-nitro- phenol absorbance. acetylcholinesterase inhibition assay was measured by 5 min incubation with a 20-fold dilution aliquot of IMP-pNP sampled from the hydrolysis reaction. Deg, Degradation; Detox, detoxification. Enzyme Time (min) % Deg (GC) %Deg (A 400 min )1 ) % Detox (acetylcholinesterase activity) Tris 1 10 5 0 V346A 1 52 57 2.5 Wild-type 1 40 52 0 Tris 10 0 4 0 V346A 10 46 63 8.0 Wild-type 10 44 53 0 Tris 60 0 5 0 V346A 60 70 82 17.5 Wild-type 60 49 53 0 Tris 180 6 5 0 V346A 180 87 98 54.5 Wild-type 180 45 53 0 Tris 420 14 5 0 V346A 420 98 100 87.4 Wild-type 420 61 53 0 G. Amitai et al. Enhanced stereoselective OP hydrolysis by PON1 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research 1913 IMP-pNP by wild-type PON1 and its single mutation variant V346A (1.2PC) at specified time intervals using both direct spectrophotometric assay and GC ⁄ PFPD analysis, used for determination of degra- dation levels of both stereoisomers. These degradation levels of IMP-pNP were compared with the levels of detoxification measured by the acetylcholinesterase inhibition assay (Table 3). Figure 8 shows the time- course of IMP-pNP detoxification as well as degrada- tion by wild-type PON1 and its variant V346A using the acetylcholinesterase inhibition assay and the spec- trophotometric method, respectively. Detoxification of IMP-pNP by the V346A PON1 variant measured by acetylcholinesterase inhibition assay fits well to a sin- gle exponential decay function (Fig. 8), whereas the time-course of p-nitrophenol release induced by V346A is biphasic (Fig. 8). A mono-exponential decay fit to the experimental detoxification data yields a sin- gle rate constant k ¼ 0.005 min )1 (r 2 ¼ 0.987). An excellent nonlinear fit (r 2 ¼ 0.999) to the experimental degradation data measured by p-nitrophenol release was obtained with the following double exponential decay function: %IMP-pNP ¼½A  expðÀk 1 tÞ þ ½B  expðÀk 2 tÞ This fit provides two rate constants k 1 ¼ 0.98 min )1 and k 2 ¼ 0.014 min )1 with almost equal spans (A ¼ 55 and B ¼ 45) consistent with equal amounts of two enantiomers in the racemic mixture. The lower rate constant of the biphasic degradation curve (k 2 ) (Fig. 8) is consistent with the first-order rate constant obtained from the acetylcholinesterase inhibition assay reflect- ing IMP-pNP detoxification (k 2 ¼ 0.014min )1 derived from the double exponential decay fit, shown by the left ordinate in Fig. 8, and k ¼ 0.005 min )1 obtained from detoxification kinetics presented on the right ordinate in Fig. 8). As shown by the spectrophotomertic and GC analy- sis, IMP-pNP was already degraded 40–52% and 52–57% by wild-type and V346A PON1, respectively, within the first minute (second and third row in the third and fourth column of Table 3, Fig. 9). In con- trast, no detoxification was observed with the V346A variant within 10 min and up to seven hours with wild-type PON1 as evidenced by the residual acetyl- cholinesterase inhibition assay (fifth column in Table 3, Fig. 9). These results are consistent with significantly faster degradation of the less toxic isomer [P(+)] of IMP-pNP compared with its more toxic stereoisomer [P(–)] by wild-type and V346A [23,24]. After 3 h in the presence of V346A, IMP-pNP was detoxified by 54% and degraded by 87–98% (third, fourth and fifth col- umn at the 11th row in Table 3, Fig. 9). Interestingly, wild-type PON1 degraded IMP-pNP only up to a level of 50% even after 21 h (Fig. 8), whereas the V346A variant caused complete degradation within 4 h (Figs 8 and 9, Table 3). This property of wild-type PON1 was utilized to enzymatically separate the more toxic P(–) stereoisomer of IMP-pNP. Racemic IMP-pNP (500 lm) was incubated with wild-type PON1 (0.1 mgÆmL )1 ) for 2 h. The enzymatic reaction was monitored spectrophotometrically by measuring the increase in the absorbance of the released p-nitrophe- nol up to the plateau level obtained at 50% degrada- tion, as demonstrated in Fig. 8. After hydrolysis by PON1, the nonhydrolyzed stereoisomer was extracted with methyl t-butyl ether. IMP-pNP concentration in methyl t-butyl ether was determined by quantitative GC analysis. The bimolecular rate constant of human acetylcholinesterase by the separated stereoisomer of IMP-pNP was k i ¼ 6.3 · 10 6 min )1 Æm )1 , which is four- fold higher than that of racemic IMP-pNP (k i ¼ 1.6 · 10 6 min )1 Æm )1 ). These results are consistent with a 16-fold difference in the rate of human acetylcholin- esterase inhibition by the P(–) compared with P(+) stereoisomer of IMP-pNP. Fig. 8. Time-course of IMP-pNP degradation and detoxification by wild-type and V346A PON1. The spectrophotometric method meas- uring the increase in A 400 of p-nitrophenol (pNP) was used for de- gradation kinetics, and the acetylcholinesterase inhibition assay was used for detoxification kinetics (50 m M Tris ⁄ HCl, pH 8, 25 °C). The left ordinate presnts the scale for residual percentage IMP-pNP during its degradation determined spectrophotometrically by p-nitro- phenol release. The right ordinate represents the percentage of putative P(–)IMP-pNP during detoxification as determined by the acetylcholinesterase inhibition assay. Enhanced stereoselective OP hydrolysis by PON1 G. Amitai et al. 1914 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research Discussion Hydrolysis of all the OPs was measured by the acetyl- cholinesterase inhibition assay. Acetylcholinesterase inhibition was measured by diluting (50–1000-fold) the intact OP remaining in solution at various time intervals during the enzymatic hydrolysis. The acetyl- cholinesterase inhibition assay is therefore sensitive to changes in concentration of the more toxic isomer of chiral OPs and reflects the rate of detoxification, rather than degradation, of cyclosarin, IMP-pNP and soman. In the case of symmetric OPs such as DFP, ChPo, paraoxon and parathiol, the acetylcholinesterase inhibi- tion assay reflects the rate of both degradation and detoxification. The rates of detoxification of soman and cyclosarin catalyzed by Ps. diminuta OPH, squid DFPase and PON1 shown in Fig. 1 were determined by calculating the initial rates of hydrolysis. The initial rate (k obs ) is equal to the slope of linear dependence of ln(% acetylcholinesterase inhibition) [parallel with ln(% residual OP)] with time. It is pertinent to note a recent report on the stereoselective hydrolysis of cyclos- arin by bacterial OPAA and OPH [30]. Hydrolysis was followed by measuring the fluoride ions released during hydrolysis. This study demonstrated a 12–24.3-fold fas- ter rate of hydrolysis by OPH and OPAA for the P(+) isomer than for the P(–) isomer. As the acetylcholinest- erase inhibition assay measures exclusively the hydroly- sis of the more toxic stereoisomer P(–)cyclosarin, the time-course profile of cyclosarin detoxification fits bet- ter a single-exponential decay (Fig. 1A) rather than a double-exponential profile, as demonstrated previously by the fluoride-release assay [30]. Possible racemization induced by fluoride ions released during hydrolysis is unlikely, as the maximal concentration of fluoride released from 10 lm cyclosarin is not sufficient for the conversion of cyclosarin enantiomers at the time scale used in our study (not shown). The slow phase of P(–)cyclosarin hydrolysis observed by Harvey et al. [30] is consistent with the slow detoxification rate of cyclos- arin by bacterial OPH measured in the present report by the acetylcholinesterase inhibition assay (k obs ¼ 2.7 · 10 )3 min )1 ; Fig. 1). Comparison of the rate of enzymatic detoxification of cyclosarin and soman using constant substrate and enzyme concentrations clearly demonstrates faster detoxification by wild-type mammalian PON1 than bac- terial OPH and squid DFPase (Fig. 1). Therefore, it was of particular interest to develop and study new PON1 variants with enhanced activity. This work describes several PON1 variants with significantly improved detoxification rates toward toxic OP substrates. Most notably, the single mutants V346A and H115W exhib- ited higher rates (11–380-fold) of hydrolysis of certain OPs compared with wild-type PON1. The newly evolved PON1 variants could be segregated into four main groups: group 1, H115W showing 270–380-fold enhanced hydrolytic activity toward the P–S bond in pa- rathiol compared with wild-type PON1 (Figs 4, 5 and 7); group 2, the single mutant L69V showing 10–100- fold enhanced activity toward P–F-containing OP com- pounds (i.e. DFP, cyclosarin and soman; Figs 2, 3 and 4); group 3, V346A, L69V ⁄ S193P ⁄ V346A and the five-site mutant L69V ⁄ S138L ⁄ S193P ⁄ N287D ⁄ V346A exhibiting a 4–10-fold higher activity toward both P–O- containing (ChPo) and P–F-containing OP esters (Fig. 4); group 4, includes the variants S193P, L69V ⁄ S193P, L69V ⁄ S138L ⁄ S193P, L69V ⁄ S138L ⁄ S193P ⁄ N287D displaying no enhancement or lower activity than wild-type PON1 toward soman and ChPo (Fig. 4). The H115W mutant is an interesting variation. His115 and His134 have been proposed as the key cat- alytic residues of PON1 [18]. However, Yeung et al. [17] have shown that the paraoxonase activity of H115W PON1 is even higher than that of the wild- Fig. 9. Time-course of IMP-pNP degradation and detoxification elici- ted by wild-type and V346A PON1 at specified time intervals pre- sented in three dimensions. Degradation of IMP-pNP was measured by GC ⁄ PFPD analysis during hydrolysis (left side of the cube: black bars, Tris buffer; red, wild-type; blue, V346A). Detoxifi- cation was monitored by residual acetylcholinesterase inhibition by IMP-pNP (right side of the cube: green bars, Tris buffer; pink, wild- type; khaki, V346A). The time axis (minutes) is drawn on a logarith- mic scale. PON1 wild-type and V346A concentration is 0.03 mgÆmL )1 ;50mM Tris, pH 8; 1 mM CaCl 2 ;25°C. G. Amitai et al. Enhanced stereoselective OP hydrolysis by PON1 FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research 1915 [...]... indicate faster hydrolysis of the less toxic stereoisomers of cyclosarin, soman and IMPpNP (Tables 2 and 3 and Figs 8 and 9) These results are consistent with those obtained by Li et al [25] with wild-type Ps diminuta OPH using p-nitrophenol analogs of soman and sarin and with biphasic stereoselective hydrolysis of cyclosarin by Alteromonas sp JD6.5 and A haloplanktis OPAA demonstrated by Harvey et al... Enzymatic degradation of IMP-pNP, paraoxon and parathiol by PON1 variants was also measured directly by monitoring the release of p-nitrophenol at 400 nm The kobs values were calculated from the linear part (r2 ¼ 0.99) of the initial rate curve corrected for spontaneous hydrolysis As Km values for a number of OP substrates are very close ($ 1 mm) and the concentrations of PON1 variants and OP substrates... identical manner [29] Recombinant serum PON1 and its directed evolution variants were produced and purified as described [19,20] Inhibition kinetics of acetylcholinesterase by the residual level of all OP compounds during enzymatic hydrolysis was performed using the Ellman method for acetylcholinesterase activity [22] Determination of detoxification activity of PON1 variants, OPH and DFPase toward OP... performed directly with the nerve agent of interest using the acetylcholinesterase inhibition assay The best variants 1916 G Amitai et al could then be further optimized by mutations at the second-shell residues identified by this study (e.g Ser138, Ser193 and Asn287), to obtain a level of catalytic efficiency that is sufficient for decontamination of these agents In summary, screening newly evolved PON1 variants, ... initial part (up to 50% OP hydrolysis) of the experimental FEBS Journal 273 (2006) 1906–1919 ª 2006 Israel Institute for Biological Research Enhanced stereoselective OP hydrolysis by PON1 nonlinear curve The kinetic experiments were performed in triplicate Non-linear fits were carried out using the GraphPad Prism program Determination of PON1 degradation activity toward OP substrates by the spectrophotometric... residues Notably, some of the OP substrates studied (e.g soman, cyclosarin and IMP-pNP) are racemic mixtures of optical stereoisomers that differ significantly in their toxicity [23–25] As the acetylcholinesterase inhibition assay measures primarily hydrolysis of the more toxic optical isomer of racemic OPs, it was pertinent to use an analytical method that would measure the degradation of all stereoisomers... close ($ 1 mm) and the concentrations of PON1 variants and OP substrates used throughout all hydrolysis experiments were equal, it is assumed that the ratio of the bimolecular rate constants of OP hydrolysis by PON1 (kcat ⁄ Km) could conceivably be represented by the ratio of kobs values, measured for a newly evolved variant compared with wild-type PON1 for each OP substrate [kobs(mutant) ⁄ kobs(wild-type)]... anticholinesterase properties, and acute toxicity in mice of four stereoisomers of the nerve agent soman Toxicol Appl Pharmacol 72, 61–74 24 Benschop HP & De Jong LPA (1988) Nerve agent stereoisomers: analysis, isolation and toxicology Acc Chem Res 21, 368–337 25 Li WS, Lum KT, Chen-Goodspeed M, Sogorb MA & Raushel FM (2001) Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase Bioorg... Biological Research Enhanced stereoselective OP hydrolysis by PON1 27 28 29 30 selectivity more effectively than distant mutations Chem Biol 12, 45–54 Khersonsky O & Tawfik DS (2005) Structure-reactivity studies of serum paraoxonase PON1 suggest that its native activity is lactonase Biochemistry 44, 6371–6382 Draganov DI, Teiber JF, Speelman A, Osawa Y, Sunahara R & La Du BN (2005) Human paraoxonases (PON1,... Cohen E, Grunwald J & Ashani Y (1998) Prophylaxis against soman inhalation toxicity in guinea pigs by pretreatment alone with human serum butyrylcholinesterase Toxicol Sci 43, 121–128 8 Cohen O, Kronman C, Chitlaru T, Ordentlich A, Velan B & Shafferman A (2001) Effect of chemical modification of recombinant human acetylcholinesterase by polyethylene glycol on its circulatory longevity Biochem J 357, 795–802 . Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase Gabriel Amitai 1 ,. and 3). Modified rates of OP detoxification by newly evolved PON1 variants The enhanced rate of detoxification of cyclosarin and soman by wild-type PON1 compared