Mononuclear binding and catalytic activity of europium(III) and gadolinium(III) at the active site of the model metalloenzyme phosphotriesterase.

Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61 Šresolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.

Tuning the Envelope Structure of Enzyme Nanoreactors for In Vivo Detoxification of Organophosphates.

Encapsulated phosphotriesterase nanoreactors show their efficacy in the prophylaxis and post-exposure treatment of poisoning by paraoxon. A new enzyme nanoreactor (E-nRs) containing an evolved multiple mutant (L72C/Y97F/Y99F/W263V/I280T) of Saccharolobus solfataricus phosphotriesterase (PTE) for in vivo detoxification of organophosphorous compounds (OP) was made. A comparison of nanoreactors made of three- and di-block copolymers was carried out. Two types of morphology nanoreactors made of di-block copolymers were prepared and characterized as spherical micelles and polymersomes with sizes of 40 nm and 100 nm, respectively. The polymer concentrations were varied from 0.1 to 0.5% (w/w) and enzyme concentrations were varied from 2.5 to 12.5 µM. In vivo experiments using E-nRs of diameter 106 nm, polydispersity 0.17, zeta-potential -8.3 mV, and loading capacity 15% showed that the detoxification efficacy against paraoxon was improved: the LD50 shift was 23.7xLD50 for prophylaxis and 8xLD50 for post-exposure treatment without behavioral alteration or functional physiological changes up to one month after injection. The pharmacokinetic profiles of i.v.-injected E-nRs made of three- and di-block copolymers were similar to the profiles of the injected free enzyme, suggesting partial enzyme encapsulation. Indeed, ELISA and Western blot analyses showed that animals developed an immune response against the enzyme. However, animals that received several injections did not develop iatrogenic symptoms.

Engineering of a phosphotriesterase with improved stability and enhanced activity for detoxification of the pesticide metabolite malaoxon.

Organophosphorus (OP) pesticides are still widely applied but pose a severe toxicological threat if misused. For in vivo detoxification, the application of hydrolytic enzymes potentially offers a promising treatment. A well-studied example is the phosphotriesterase of Brevundimonas diminuta (BdPTE). Whereas wild-type BdPTE can hydrolyse pesticides like paraoxon, chlorpyrifos-oxon and mevinphos with high catalytic efficiencies, kcat/KM >2 × 107 M-1 min-1, degradation of malaoxon is unsatisfactory (kcat/KM ≈ 1 × 104 M-1 min-1). Here, we report the rational engineering of BdPTE mutants with improved properties and their efficient production in Escherichia coli. As result, the mutant BdPTE(VRNVVLARY) exhibits 37-fold faster malaoxon hydrolysis (kcat/KM = 4.6 × 105 M-1 min-1), together with enhanced expression yield, improved thermal stability and reduced susceptibility to oxidation. Therefore, this BdPTE mutant constitutes a powerful candidate to develop a biocatalytic antidote for the detoxification of this common pesticide metabolite as well as related OP compounds.

The impact of molecular variants, crystallization conditions and the space group on ligand-protein complexes: a case study on bacterial phosphotriesterase.

A bacterial phosphotriesterase was employed as an experimental paradigm to examine the effects of multiple factors, such as the molecular constructs, the ligands used during protein expression and purification, the crystallization conditions and the space group, on the visualization of molecular complexes of ligands with a target enzyme. In this case, the ligands used were organophosphates that are fragments of the nerve agents and insecticides on which the enzyme acts as a bioscavenger. 12 crystal structures of various phosphotriesterase constructs obtained by directed evolution were analyzed, with resolutions of up to 1.38 Å. Both apo forms and holo forms, complexed with the organophosphate ligands, were studied. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing the organophosphate complexes of the enzyme. The study revealed that the tags used for protein expression can lodge in the active site and hinder ligand binding. Furthermore, the space group in which the protein crystallizes can significantly impact the visualization of bound ligands. It was also observed that the crystallization precipitants can compete with, and even preclude, ligand binding, leading to false positives or to the incorrect identification of lead drug candidates. One of the co-crystallization conditions enabled the definition of the spaces that accommodate the substituents attached to the P atom of several products of organophosphate substrates after detachment of the leaving group. The crystal structures of the complexes of phosphotriesterase with the organophosphate products reveal similar short interaction distances of the two partially charged O atoms of the P-O bonds with the exposed ß-Zn2+ ion and the buried α-Zn2+ ion. This suggests that both Zn2+ ions have a role in stabilizing the transition state for substrate hydrolysis. Overall, this study provides valuable insights into the challenges and considerations involved in studying the crystal structures of ligand-protein complexes, highlighting the importance of careful experimental design and rigorous data analysis in ensuring the accuracy and reliability of the resulting phosphotriesterase-organophosphate structures.

A mesophilic phosphotriesterase-like lactonase shows high stability and proficiency as quorum quenching enzyme.

The problem of biofilm formation is a serious concern under various pathological conditions such as extensive burns, wounds in diabetic patients, bedsores, cystic fibrosis, nosocomial infections from implantable medical devices such as catheters, valves, etc. Environmental diffusion of biofilm (in pools, wet floors, industrial food plants) that could represent a reservoir of antibiotic resistant bacteria constitues an additional issue. In this work is described a lactonase from Rhodococcus erythropolis, a phosphotriesterase-like lactonase (PLL) enzyme, which has already been studied in the past and can be used for containment of biofilm formation. The protein is 28% and 40% identical with respect to the Pseudomonas diminuta PTE and the thermostable Saccharolobus solfataricus SsoPox respectively. The protein was obtained starting from a synthetic His-tagged gene, expressed in E. coli, purified and further characterized. New properties, not previously known or deducible from its sequence, have been highlighted. These properties are: the enzyme is thermophilic and thermostable even though it originates from a mesophilic bacterium; the enzyme has a long (months) shelf life at 4 °C; the enzyme is not only stable to low concentrations of the oxidant H2O2 but even activated by it at high concentrations; the enzyme proved to be a proficient quorum quenching enzyme, able to hydrolase acyl-homoserine lactones 3oxoC12-HSL and C4-HSL, and can inhibit up to 60% the formation of Pseudomonas aeruginosa (PAO1) biofilm. These different properties make the lactonase useful to fight resistant bacteria that induce inflammatory and infectious processes mediated by the quorum sensing mechanism.

QM/MM and MM MD Simulations on Enzymatic Degradation of the Nerve Agent VR by Phosphotriesterase.

V-type nerve agents are hardly degraded by phosphotriesterase (PTE). Interestingly, the PTE variant of BHR-73MNW can effectively improve the hydrolytic efficiency of VR, especially for its Sp-enantiomer. Here, the whole enzymatic degradation of both Sp and Rp enantiomers of VR by the wild-type PTE and its variant BHR-73MNW was investigated by quantum mechanics/molecular mechanics (QM/MM) calculations and MM molecular dynamics simulations. Present results indicate that the degradation of VR can be initiated by the nucleophilic attack of the bridging OH- and the zinc-bound water molecule. The QM/MM-predicted energy barriers for the hydrolytic process of Sp-VR are 19.8 kcal mol-1 by the variant with water as a nucleophile and 22.0 kcal mol-1 by the wild-type PTE with OH- as a nucleophile, and corresponding degraded products are bound to the dinuclear metal site in monodentate and bidentate coordination modes, respectively. The variant effectively increases the volume of the large pocket, allowing more water molecules to enter the active pocket and resulting in the improvement of the degradation efficiency of Sp-VR. The hydrolysis of Rp-VR is triggered only by the hydroxide with an energy span of 20.6 kcal mol-1 for the wild-type PTE and 20.7 kcal mol-1 for the variant BHR-73-MNW PTE. Such mechanistic insights into the stereoselective degradation of VR by PTE and the role of water may inspire further studies to improve the catalytic efficiency of PTE toward the detoxification of nerve agents.

Structural and Functional Analysis of a Highly Active Designed Phosphotriesterase for the Detoxification of Organophosphate Nerve Agents Reveals an Unpredicted Conformation of the Active Site Loop.

Neurotoxic organophosphorus compounds (OPs) pose a severe threat if misused in military conflicts or by terrorists. Administration of a hydrolytic enzyme that can decompose the circulating nerve agent into non-toxic metabolites in vivo offers a potential treatment. A promising candidate is the homo-dimeric phosphotriesterase originating from the bacterium Brevundimonas diminuta (BdPTE), which has been subject to several rational and combinatorial protein design studies. A series of engineered versions with much improved catalytic efficiencies toward medically relevant nerve agents was described, carrying up to 22 mutations per enzyme subunit. To provide a basis for further rational design, we have determined the crystal structure of the highly active variant 10-2-C3(C59V/C227V)─stabilized against oxidation by substitution of two unpaired Cys residues─in complex with a substrate analogue at 1.5 Å resolution. Unexpectedly, the long loop segment (residues 253-276) that covers the active site shows a totally new conformation, with drastic structural deviations up to 19 Å, which was neither predicted in any of the preceding protein design studies nor seen in previous crystallographic analyses of less far evolved enzyme versions. Inspired by this structural insight, additional amino acid exchanges were introduced and their effects on protein stability as well as on the catalytic efficiency toward several neurotoxic OPs were investigated. Somewhat surprisingly, our results suggest that the presently available engineered version of BdPTE, in spite of its design on the basis of partly false structural assumptions, constitutes a fairly optimized enzyme for the detoxification of relevant OP nerve agents.

Backbone and side chain chemical shift assignment of diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris, an organophosphorus-degrading enzyme.

NMR chemical shift assignments are reported for backbone (15N, 1H) and partial side chain (13Cα and ß, side chain 1H) atoms of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of hydrolyzing phosphorus - fluorine bonds in a variety of toxic organophosphorus compounds. Analysis of residues lining the active site of DFPase highlight a number of residues whose chemical shifts can be used as a diagnostic of binding and detection of organophosphorus compounds.

Ultrahigh-Throughput Directed Evolution of a Metal-Free α/ß-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase.

Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91's activity by a factor of ≈360 (to a kcat/KM of ≈7 × 105 M-1 s-1) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries.

Immobilization of a Broad Range of Polypeptides on the Frustule of the Diatom Thalassiosira pseudonana.

Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. One recently developed technique, living diatom silica immobilization (LiDSI), has made it possible to immobilize proteins, including multimeric and redox enzymes, via a cellular excretion system onto the silica frustule of the marine diatom Thalassiosira pseudonana. However, the number of application examples so far is limited, and the type of proteins appropriate for the technique is still enigmatic. Here, we applied LiDSI to six industrially relevant polypeptides, including protamine, metallothionein, phosphotriesterase, choline oxidase, laccase, and polyamine synthase. Protamine and metallothionein were successfully immobilized on the frustule as protein fusions with green fluorescent protein (GFP) at the N terminus, indicating that LiDSI can be used for polypeptides which are rich in arginine and cysteine. In contrast, we obtained mutants for the latter four enzymes in forms without green fluorescent protein. Immobilized phosphotriesterase, choline oxidase, and laccase showed enzyme activities even after the purification of frustule in the presence of 1% (wt/vol) octylphenoxy poly(ethyleneoxy)ethanol. An immobilized branched-chain polyamine synthase changed the intracellular polyamine composition and silica nanomorphology. These results illustrate the possibility of LiDSI for industrial applications. IMPORTANCE Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. Living diatom silica immobilization (LiDSI) is a recently developed technique for in vivo protein immobilization on the diatom frustule. We aimed to explore the possibility of using LiDSI for industrial applications by successfully immobilizing six polypeptides: (i) protamine (Oncorhynchus keta), a stable antibacterial agent; (ii) metallothionein (Saccharomyces cerevisiae), a metal adsorption molecule useful for bioremediation; (iii) phosphotriesterase (Sulfolobus solfataricus), a scavenger for toxic organic phosphates; (iv) choline oxidase (Arthrobacter globiformis), an enhancer for photosynthetic activity and yield of plants; (v) laccase (Bacillus subtilis), a phenol oxidase utilized for delignification of lignocellulosic materials; and (vi) branched-chain polyamine synthase (Thermococcus kodakarensis), which produces branched-chain polyamines important for DNA and RNA stabilization at high temperatures. This study provides new insights into the field of applied biological materials.

Characterization of the phosphotriesterase capable of hydrolyzing aryl-organophosphate flame retardants.

A related group of phosphotriesters known as organophosphate flame retardants (OPFRs) has become emerging contaminants due to its worldwide use. The lack of an easily hydrolysable bond renders OPFRs inert to the well-known phosphotriesterases capable of hydrolyzing the neurotoxic organophosphates. An OPFRs phosphotriesterase gene stpte was cloned from plasmid pStJH of strain Sphingopyxis terrae subsp. terrae YC-JH3 and was heterologously expressed in Escherichia coli. The recombinant protein St-PTE was purified and analyzed. St-PTE showed the highest catalytic activity at pH 8.5 and 35 °C. The optimal substrate for St-PTE is triphenyl phosphate, with kcat/Km of 5.03 × 106 M-1 s-1, two orders of magnitude higher than those of tricresyl phosphate (4.17 × 104 M-1 s-1), 2-ethylhexyl diphenyl phosphate (2.03 × 104 M-1 s-1) and resorcinol bis(diphenyl phosphate) (6.30 × 104 M-1 s-1). St-PTE could break the P-O bond of tri-esters and convert aryl-OPFRs into their corresponding di-ester metabolites, including polymers of resorcinol bis(diphenyl phosphate). Mediated by transposase, the gene of OPFRs phosphotriesterase could be transferred horizontally among closely related strains of Sphingomonas, Sphingobium and Sphingopyxis. KEY POINTS: • St-PTE from Sphingopyxis terrae subsp. terrae YC-JH3 could hydrolyze aryl-OPFRs. • Metabolites of RBDPP hydrolyzed by phosphotriesterase were identified. • St-PTE could hydrolyze the P-O cleavage of dimer and trimer of RBDPP. • Phosphotriesterase genes transfer among Sphingomonadaceae mediated by transposase.

Utilization of diverse organophosphorus pollutants by marine bacteria.

Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Water-Regulated Mechanisms for Degradation of Pesticides Paraoxon and Parathion by Phosphotriesterase: Insight from QM/MM and MD Simulations.

The enzymatic degradation of pesticides paraoxon (PON) and parathion (PIN) by phosphotriesterase (PTE) has been investigated by QM/MM calculations and MD simulations. In the PTE-PON complex, Znα and Znß in the active site are five- and six-coordinated, respectively, while both zinc ions are six coordinated with the Znα -bound water molecule (WT1) for the PTE-PIN system. The hydrolytic reactions for PON and PIN are respectively driven by the nucleophilic attack of the bridging-OH- and the Znα -bound water molecule on the phosphorus center of substrate, and the two-step hydrolytic process is predicted to be the rate-limiting step with the energy spans of 13.8 and 14.4 kcal/mol for PON and PIN, respectively. The computational studies reveal that the presence of the Znα -bound water molecule depends on the structural feature of substrates characterized by P=O and P=S, which determines the hydrolytic mechanism and efficiency for the degradation of organophosphorus pesticides by PTE.

QM/MM and MM MD simulations on decontamination of the V-type nerve agent VX by phosphotriesterase: toward a comprehensive understanding of steroselectivity and activity.

Due to deadly toxicity and high environmental stability of the nerve agent VX, an efficient decontamination approach is desperately needed in tackling its severe threat to human security. The enzymatic destruction of nerve agents has been generally considered as one of the most effective ways, and here the hydrolysis of VX by phosphotriesterase (PTE) was investigated by extensive QM/MM and MM MD simulations. The hydrolytic cleavage of P-S by PTE is a two-step process with the free energy spans of 15.8 and 26.0 kcal mol-1 for the RP- and SP-enantiomer VX, respectively, and such remarkable stereospecificity of VX enantiomers in the enzymatic degradation is attributed to their conformational compatibility with the active pocket. The structurally less adaptive SP-enantiomer allows one additional water molecule to enter the binuclear zinc center and remarkably facilitates the release of the degraded product. Overall, the rate-limiting steps in the enzymatic degradation of VX by PTE involve the degraded product release of the RP-enantiomer and the enzymatic P-S cleavage of the SP-enantiomer. Further computational analysis on the mutation of selected residues also revealed that H257Y, H257D, H254Q-H257F, and L7ep-3a variants allow more water molecules to enter the active site, which improves the catalytic efficiency of PTE, as observed experimentally. The present work provides mechanistic insights into the stereoselective hydrolysis of VX by PTE and the activity manipulation through the active-site accessibility of water molecules, which can be used for the enzyme engineering to defeat chemical warfare agents.

Enzyme Nanoreactor for In Vivo Detoxification of Organophosphates.

A nanoreactor containing an evolved mutant of Saccharolobus solfataricus phosphotriesterase (L72C/Y97F/Y99F/W263V/I280T) as a catalytic bioscavenger was made for detoxification of organophosphates. This nanoreactor intended for treatment of organophosphate poisoning was studied against paraoxon (POX). Nanoreactors were low polydispersity polymersomes containing a high concentration of enzyme (20 µM). The polyethylene glycol-polypropylene sulfide membrane allowed for penetration of POX and exit of hydrolysis products. In vitro simulations under second order conditions showed that 1 µM enzyme inactivates 5 µM POX in less than 10 s. LD50-shift experiments of POX-challenged mice through intraperitoneal (i.p.) and subcutaneous (s.c.) injections showed that intravenous administration of nanoreactors (1.6 nmol enzyme) protected against 7 × LD50 i.p. in prophylaxis and 3.3 × LD50 i.p. in post-exposure treatment. For mice s.c.-challenged, LD50 shifts were more pronounced: 16.6 × LD50 in prophylaxis and 9.8 × LD50 in post-exposure treatment. Rotarod tests showed that transitory impaired neuromuscular functions of challenged mice were restored the day of experiments. No deterioration was observed in the following days and weeks. The high therapeutic index provided by prophylactic administration of enzyme nanoreactors suggests that no other drugs are needed for protection against acute POX toxicity. For post-exposure treatment, co-administration of classical drugs would certainly have beneficial effects against transient incapacitation.

Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.

Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid Loligo vulgaris and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 °C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.

Degradation of pesticides diazinon and diazoxon by phosphotriesterase: insight into divergent mechanisms from QM/MM and MD simulations.

Enzymatic hydrolysis by phosphotriesterase (PTE) is one of the most effective ways of degrading organophosphorus pesticides, but the catalytic efficiency depends on the structural features of substrates. Here the enzymatic degradation of diazinon (DIN) and diazoxon (DON), characterized by PS and PO, respectively, have been investigated by QM/MM calculations and MM MD simulations. Our calculations demonstrate that the hydrolysis of DON (with PO) is inevitably initiated by the nucleophilic attack of the bridging-OH- on the phosphorus center, while for DIN (with PS), we proposed a new degradation mechanism, initiated by the nucleophilic attack of the Znα-bound water molecule, for its low-energy pathway. For both DIN and DON, the hydrolytic reaction is predicted to be the rate-limiting step, with energy barriers of 18.5 and 17.7 kcal mol-1, respectively. The transportation of substrates to the active site, the release of the leaving group and the degraded product are generally verified to be favorable by MD simulations via umbrella sampling, both thermodynamically and dynamically. The side-chain residues Phe132, Leu271 and Tyr309 play the gate-switching role to manipulate substrate delivery and product release. In comparison with the DON-enzyme system, the degraded product of DIN is more easily released from the active site. These new findings will contribute to the comprehensive understanding of the enzymatic degradation of toxic organophosphorus compounds by PTE.

Post-VX exposure treatment of rats with engineered phosphotriesterases.

The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M-1 min-1) towards VX and a preferential hydrolysis of the more toxic P(-) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg-1 i.v.: PTE-2 dosed at 1.3 mg kg-1 i.v. (PTE-2.1) and 2.6 mg kg-1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg-1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.

A Rationally Designed Supercharged Protein-Enzyme Chimera Self-Assembles In Situ to Yield Bifunctional Composite Textiles.

Catalytically active materials for the enhancement of personalized protective equipment (PPE) could be advantageous to help alleviate threats posed by neurotoxic organophosphorus compounds (OPs). Accordingly, a chimeric protein comprised of a supercharged green fluorescent protein (scGFP) and phosphotriesterase from Agrobacterium radiobacter (arPTE) was designed to drive the polymer surfactant (S-)-mediated self-assembly of microclusters to produce robust, enzymatically active materials. The chimera scGFP-arPTE was structurally characterized via circular dichroism spectroscopy and synchrotron radiation small-angle X-ray scattering, and its biophysical properties were determined. Significantly, the chimera exhibited greater thermal stability than the native constituent proteins, as well as a higher catalytic turnover number (kcat). Furthermore, scGFP-arPTE was electrostatically complexed with monomeric S-, driving self-assembly into [scGFP-arPTE][S-] nanoclusters, which could be dehydrated and cross-linked to yield enzymatically active [scGFP-arPTE][S-] porous films with a high-order structure. Moreover, these clusters could self-assemble within cotton fibers to generate active composite textiles without the need for the pretreatment of the fabrics. Significantly, the resulting materials maintained the biophysical activities of both constituent proteins and displayed recyclable and persistent activity against the nerve agent simulant paraoxon.

Probing the Suitability of Different Ca2+ Parameters for Long Simulations of Diisopropyl Fluorophosphatase.

Organophosphate hydrolases are promising as potential biotherapeutic agents to treat poisoning with pesticides or nerve gases. However, these enzymes often need to be further engineered in order to become useful in practice. One example of such enhancement is the alteration of enantioselectivity of diisopropyl fluorophosphatase (DFPase). Molecular modeling techniques offer a unique opportunity to address this task rationally by providing a physical description of the substrate-binding process. However, DFPase is a metalloenzyme, and correct modeling of metal cations is a challenging task generally coming with a tradeoff between simulation speed and accuracy. Here, we probe several molecular mechanical parameter combinations for their ability to empower long simulations needed to achieve a quantitative description of substrate binding. We demonstrate that a combination of the Amber19sb force field with the recently developed 12-6 Ca2+ models allows us to both correctly model DFPase and obtain new insights into the DFP binding process.