Mutants Of Phosphotriesterase Research Articles

An Enzyme-Based Biosensor for the Detection of Organophosphate Compounds Using Mutant Phosphotriesterase Immobilized onto Reduced Graphene Oxide

Enzymatic detection of organophosphate (OP) compounds can be tailored using highly sensitive and selective enzymes in the development of biosensors. Previously, mutant (YT) phosphotriesterase (PTE) was reported to efficiently hydrolyze Sp and Rp enantiomers of phosphotriester. This study reports the use of phosphotriesterase mutant YT (YT-PTE) immobilized onto reduced graphene oxide (rGO) and fabricated onto a screen-printed carbon electrode (SPCE) for electrochemical detection of OP compounds. Immobilization of YT-PTE onto rGO was secured using N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) cross-linker, and the resulting immobilized enzyme was able to retain up to 90% of its activity. Electrochemical analysis of the SPCE/rGO/YT-PTE showed detection of paraoxon in a linear range of 1 mM–0.005 μM with its limit of detection as low as 0.11 μM. SPCE/rGO/YT-PTE exhibited high selectivity towards paraoxon and parathion and have good reproducibility. Furthermore, detection of paraoxon was also possible in a real water sample with only minor interferences.

Theoretical Study of VX Hydrolysis Mechanism Catalyzed by Phosphotriesterase Mutant H254R

AbstractPhosphotriesterase (PTE) is a bacterial enzyme that can catalyze the hydrolysis of V‐type chemical warfare nerve agents. Herein, the hydrolysis mechanism of VX analogs catalyzed by the H254R PTE mutant, in which the histidine 254 residue is replaced by an arginine, has been investigated using the hybrid density functional theory method B3LYP. The chemical model of the active site of H254R PTE mutant was constructed based on the high resolution X‐ray crystal structure. We have presented the energetics and the characterization of stationary points in the catalytic reaction. The calculations indicate that the catalytic process contain two SN2 displacement steps and the step of hydroxide regeneration is rate‐limiting with an overall barrier of 22.2 kcal/mol. The Arg254 plays an important role as a proton donor/acceptor for protonating leaving group and activating water molecule. Moreover, the H254R PTE mutant with binuclear cobalt center turns out to higher activity towards VX than the binuclear zinc H254R PTE.

Enzyme-Catalyzed Kinetic Resolution of Chiral Precursors to Antiviral Prodrugs.

Nucleoside analogues are among the most common medications given for the treatment of viral infections and cancers. The therapeutic effectiveness of nucleoside analogues can be dramatically improved by phosphorylation. The ProTide approach was developed using a phosphorylated nucleoside that is masked by esterification with an amino acid and phenol forming a chiral phosphorus center. The biological activity of the ProTides depends, in part, on the stereochemistry at phosphorus, and thus, it is imperative that efficient methods be developed for the chemical synthesis and isolation of diastereomerically pure ProTides. Chiral ProTides are often synthesized by direct displacement of a labile phenol (p-nitrophenol or pentafluorophenol) from a chiral phosphoramidate precursor with the appropriate nucleoside analogue. The ability to produce these chiral products is dictated by the synthesis of the chiral phosphoramidate precursors. The enzyme phosphotriesterase (PTE) from Pseudomonas diminuta is well-known for its high stereoselectivity and broad substrate profile. Screening PTE variants from enzyme evolution libraries enabled the identification of variants of PTE that can stereoselectively hydrolyze the chiral phosphoramidate precursors. The variant G60A-PTE exhibits a 165-fold preference for hydrolysis of the RP isomer, while the variant In1W-PTE has a 1400-fold preference for hydrolysis of the SP isomer. Using these mutants of PTE, the SP and RP isomers were isolated on a preparative scale with no detectable contamination of the opposite isomer. Combining the simplicity of the enzymatic resolution of the precursor with the latest synthetic strategy will facilitate the production of diastereometrically pure nucleotide phosphoramidate prodrugs.

An OPAA enzyme mutant with increased catalytic efficiency on the nerve agents sarin, soman, and GP.

The wild-type OPAA enzyme has relatively high levels of catalytic activity against several organophosphate G-type nerve agents. A series of mutants containing replacement amino acids at the OPAA Y212, V342, and I215 sites showed several fold enhanced catalytic efficiency on sarin, soman, and GP. One mutant, Y212F/V342L, showed enhanced stereospecificity on sarin and that enzyme along with a phosphotriesterase mutant, GWT, which had the opposite stereospecificity, were used to generate enriched preparations of each sarin enantiomer. Inhibition of acetylcholinesterase by the respective enantioenriched sarin solutions subsequently provided identification of the sarin enantiomers as separated by normal phase enantioselective liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry.

Post-exposure treatment of VX poisoned guinea pigs with the engineered phosphotriesterase mutant C23: A proof-of-concept study

The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(−) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (kcat/KM=5×106M−1min−1). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5mgkg−1 i.v.) 5min after s.c. challenge with VX (∼2LD50) prevented systemic toxicity. A lower C23 dose (2mgkg−1) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15min post VX) with 5mgkg−1 C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(−)-VX enantiomer that correlates with the C23 blood levels and its kcat/KM value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single post-exposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication.

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

The V-type nerve agents (VX and VR) are among the most toxic substances known. The high toxicity and environmental persistence of VX make 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 12 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 diethoxyphosphate core rather than the ethoxymethylphosphonate core of VX. The evolved catalytic activity with DEVX was enhanced 26-fold relative to wild-type 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 = 7 × 10(4) M(-1) s(-1), a 230-fold improvement relative to 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.

Structural Determinants for the Stereoselective Hydrolysis of Chiral Substrates by Phosphotriesterase

Wild-type phosphotriesterase (PTE) preferentially hydrolyzes the R(P) enantiomers of the nerve agents sarin (GB) and cyclosarin (GF) and their chromophoric analogues. The active site of PTE can be subdivided into three binding pockets that have been denoted as the small, large, and leaving group pockets based on high-resolution crystal structures. The sizes and shapes of these pockets dictate the substrate specificity and stereoselectivity for catalysis. Mutants of PTE that exhibit substantial changes in substrate specificity and the ability to differentiate between chiral substrates have been prepared. For example, the G60A mutant is stereoselective for the hydrolysis of the R(P) enantiomer of the chromophoric analogues of sarin and cyclosarin, whereas the H254G/H257W/L303T (GWT) mutant reverses the stereoselectivity for the enantiomers of these two compounds. Molecular dynamics simulations and high-resolution X-ray structures identified the correlations between structural changes in the active site and the experimentally determined kinetic parameters for substrate hydrolysis. New high-resolution structures were determined for the H257Y/L303T (YT), I106G/F132G/H257Y (GGY), and H254Q/H257F (QF) mutants of PTE. Molecular dynamics calculations were conducted using the S(P) and R(P) enantiomers of the analogues for sarin and cyclosarin for the wild-type PTE and the G60A, YT, GGY, QF, and GWT mutants. The experimental stereoselectivity correlated nicely with the difference in the computed angle of attack for the nucleophilic hydroxide relative to the phenolic leaving group of the substrate.

Stereospecificity in the enzymatic hydrolysis of cyclosarin (GF)

Enzymatic catalysis is one means of accelerating the rate of hydrolysis of G-type organophosphorus nerve agents. Here, the stereospecificity of the catalysis of cyclosarin (GF, O-cyclohexyl methylphosphonofluoridate) hydrolysis by several enzymes was investigated. Stereospecificity was not evident at 3 mM GF but was evident at 0.5 mM GF. The differential effect was apparently due to fluoride-catalyzed racemization of the substrate. Alteromonas sp. JD6.5 organophosphorus acid anhydrolase (OPAA), Alteromonas haloplanktis OPAA and the wild-type phosphotriesterase (PTE) enzymes were all found to catalyze preferentially the hydrolysis of the (+)GF isomer, as determined by GC analysis of the remaining unreacted (−)GF isomer. Acetylcholinesterase inhibition experiments showed the purified (−)GF isomer to be approximately twice as toxic as the racemic mixture. One PTE mutant, H254G/H259W/L303T, was found to reverse the native PTE stereospecificity and preferentially catalyze the hydrolysis of the (−)GF isomer, as shown by its complementation of Alteromonas sp. JD6.5 OPAA and by GC analysis of the remaining (+)GF isomer. This procedure also permitted the individual preparation of either of the two GF isomers by enzymatic degradation followed by extraction of the remaining isomer.

Increased expression of a bacterial phosphotriesterase in Escherichia coli through directed evolution

We devised a growth-based strategy for screening phosphotriesterase mutant libraries for variants with enhanced activity towards organophosphates that generate dimethyl phosphate when hydrolysed. Phosphotriesterase mutants were screened for activity by growing transformed Escherichia coli on agar plates containing methyl paraoxon as a sole phosphorus source. E. coli is capable of growth under these conditions when coexpressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) and the glycerophosphodiester phosphodiesterase from Enterobacter aerogenes (GpdQ). The latter enzyme can hydrolyse the dimethyl phosphate produced by the phosphotriesterase to methyl phosphate, which can then be used by E. coli as a source of phosphate. Phosphotriesterase was expressed from the lac promoter at levels such that its activity was growth-rate limiting. Cultures of the largest colonies (1% of the transformants) were assayed for activity towards paraoxon spectrophotometrically in microtitre plates. This process produced E. coli variants with higher whole cell activity than wild-type, which was found to be a consequence of increased protein expression rather than any increase in enzymatic activity. The mutations present in these mutant enzymes with increased expression were exclusively in the coding region, suggesting the improvement occurs post-transcriptionally.

Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence.

Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with Km and kcat respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T < or =35 degrees C, followed by an activation phase near 45-48 degrees C and then inactivation which was completed at 60 degrees C. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different T (m) values of 60.5-67 degrees C, 58-64 degrees C and 53-64 degrees C respectively). Requirements for optimum activity of PTE (displayed by Co2+-PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated.

The wild type bacterial Co 2+/Co 2+-phosphotriesterase shows a middle-range thermostability

The phosphotriesterase (PTE) from Pseudomonas diminuta, a metalloenzyme that catalyses the hydrolysis of organophosphorus pesticides and nerve agents, has been described as a remarkably heat-stable protein [Grimsley et al., Biochemistry 36 (1997), 14366–14374]. Because substitution of the naturally occurring zinc ions by cobalt ions was found to enhance the enzyme catalytic activity, we investigated the thermal stability of the Co 2+/Co 2+-PTE. This study, carried out using capillary electrophoresis under optimised conditions in the pH range 9–10 compatible with optimal enzyme activity, provided evidence for irreversible denaturation according to the Lumry–Eyring model. A temperature-induced conformational transition ( T m≈58°C) and an early growing of aggregates were observed. Comparison of UV spectra with heat-induced inactivation data clearly demonstrated that the PTE state populated above T m was neither native nor active. Differential scanning calorimetry showed only an exothermic trace due to aggregation of the denatured protein at T=76°C. Accordingly, the temperature-induced denaturation process of the PTE could be described by a consecutive reaction model, including formation of an intermediate with enhanced activity at T≈45°C and an inactive unfolded state populated at T≈58°C, which leads to denatured aggregates. Thus, the wild type Co 2+/Co 2+-PTE displays a middle-range thermostability. Hence, for decontamination purposes under extreme Earth temperatures, wild type and engineered mutants of PTE substituted with other metal cations should be evaluated.

Stereoselective Detoxification of Chiral Sarin and Soman Analogues by Phosphotriesterase

The catalytic activity of the bacterial phosphotriesterase (PTE) toward a series of chiral analogues of the chemical warfare agents sarin and soman was measured. Chemical procedures were developed for the chiral syntheses of the S P- and R P-enantiomers of O-isopropyl p-nitrophenyl methylphosphonate (sarin analogue) in high enantiomeric excess. The R P-enantiomer of the sarin analogue ( k cat=2600 s −1) was the preferred substrate for the wild-type PTE relative to the corresponding S P-enantiomer ( k cat=290 s −1). The observed stereoselectivity was reversed using the PTE mutant, I106A/F132A/H254Y where the k cat values for the R P- and S P-enantiomers were 410 and 4200 s −1, respectively. A chemo-enzymatic procedure was developed for the chiral synthesis of the four stereoisomers of O-pinacolyl p-nitrophenyl methylphosphonate (soman analogue) with high diastereomeric excess. The R P R C-stereoisomer of the soman analogue was the preferred substrate for PTE. The k cat values for the soman analogues were measured as follows: R P R C, 48 s −1; R P S C, 4.8 s −1; S P R C, 0.3 s −1, and S P S C, 0.04 s −1. With the I106A/F132A/H254Y mutant of PTE the stereoselectivity toward the chiral phosphorus center was reversed. With the triple mutant the k cat values for the soman analogues were found to be as follows: R P R C, 0.3 s −1; R P S C, 0.3 s −1; S P R C, 11 s −1, and S P S C, 2.1 s −1. Prior investigations have demonstrated that the S P-enantiomers of sarin and soman are significantly more toxic than the R P-enantiomers. This investigation has demonstrated that mutants of the wild-type PTE can be readily constructed with enhanced catalytic activities toward the most toxic stereoisomers of sarin and soman.

ChemInform Abstract: Rationally Engineered Mutants of Phosphotriesterase for Preparative Scale Isolation of Chiral Organophosphates.

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Rationally Engineered Mutants of Phosphotriesterase for Preparative Scale Isolation of Chiral Organophosphates

will catalyze the hydrolysis of the most acidic phenolic substituent from an organophosphate triester. The overall rate of hydrolysis is very much dependent on the pKa of the leaving group phenol. 4b To enhance the substrate stereoselectivity exhibited by the wildtype enzyme, we designed and characterized several site-directed mutants of PTE. Three regions within the active site of PTE (small, large, and leaVing group) have been identified that interact with the three substituents attached to the phosphorus center of typical substrates. 7 These subsites were graphically localized using the X-ray crystal structure of PTE in the presence of a bound substrate analogue. The relative sizes of these binding subsites appear to play the dominant roles in establishing the stereoselective properties of the wild-type PTE. 8 Thus, when the small subsite is reduced in size by mutating Gly60 to an alanine residue, the observed stereoselectivity of the mutant enzyme is dramatically enhanced toward the hydrolysis of the SP-isomer. For the racemic pairs of substrates I, II, and III, the kcat/Km values are 10- to 400-fold greater for the SP-isomers than for the corresponding RP-isomers and 10 000- to 15 000-fold greater for the

A combinatorial library for the binuclear metal center of bacterial phosphotriesterase.

Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had < 10(-5) of wild-type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon.

Augmented Hydrolysis of Diisopropyl Fluorophosphate in Engineered Mutants of Phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. The rate of hydrolysis depends on the substrate and can range from 6000 s-1 for paraoxon to 0.03 s-1 for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active site residues in the wild type protein to a bound substrate analog were measured, and Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group. Eleven mutants were created using site-directed mutagenesis and purified to homogeneity. Phe132 and Phe306 were replaced by tyrosine and/or histidine to generate all combinations of single and double mutants at these two sites. The single mutants W131K, F306K, and F306E were also constructed. Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. Vmax values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant. There were significant increases in the Km for paraoxon for all mutants except F132H. Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold. Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K. Km values for acephate ranged from near wild type to a 5-fold increase. Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The Vmax value for the F132H/F306H mutant was increased to 3100 s-1. These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.