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The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, Arthrobacter sp. 4Hβ as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining ∼50% activity after 132 days since purification. Next, the addition of Arthrobacter sp. 4Hβ, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.

Phosphotriesterases (PTEs) represent a class of enzymes capable of efficient neutralization of organophosphates (OPs), a dangerous class of neurotoxic chemicals. PTEs suffer from low catalytic activity, particularly at higher temperatures, due to low thermostability and low solubility. Supercharging, a protein engineering approach via selective mutation of surface residues to charged residues, has been successfully employed to generate proteins with increased solubility and thermostability by promoting charge-charge repulsion between proteins. We set out to overcome the challenges in improving PTE activity against OPs by employing a computational protein supercharging algorithm in Rosetta. Here, we discover two supercharged PTE variants, one negatively supercharged (with -14 net charge) and one positively supercharged (with +12 net charge) and characterize them for their thermodynamic stability and catalytic activity. We find that positively supercharged PTE possesses slight but significant losses in thermostability, which correlates to losses in catalytic efficiency at all temperatures, whereas negatively supercharged PTE possesses increased catalytic activity across 25°C-55°C while offering similar thermostability characteristic to the parent PTE. The impact of supercharging on catalytic efficiency will inform the design of shelf-stable PTE and criteria for enzyme engineering., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Parathion, a once commonly used pesticide known for its potential toxicity, can follow several degradation mechanisms in the environment. Given the species stability and persistence, parathion can be washed into waterways from rain, and therefore an atomistic perspective of the hydrolysis of parathion, and its byproduct paraoxon, is required in order to understand its fate in the environment. Experimental studies have determined that pH plays an important role in the calculated hydrolysis rate constants of parathion degradation. In this work, the degradation of parathion into either paraoxon or 4-nitrophenol, and the degradation of paraoxon to 4-nitrophenol are explored through density functional theory using the M06-2X functional. How the level of basicity affects the reaction mechanism is explored through two different hydroxide/water environments. Our calculations support the anticipated mechanisms determined by previous experimental work that the formation of 4-nitrophenol is the predominant pathway in hydrolysis of parathion.

Exposure to organophosphorus pesticides (OP) can have chronic adverse effects that are independent of inhibition of acetylcholinesterase, the classic target for acute OP toxicity. In pure proteins, the organophosphorus pesticide chlorpyrifos oxon induces a cross-link between lysine and glutamate (or aspartate) with loss of water. Tubulin is particularly sensitive to OP-induced cross-linking. Our goal was to explore OP-induced cross-linking in a complex protein sample, MAP-rich tubulin from Sus scrofa and to test 8 OP for their capacity to promote isopeptide cross-linking. We treated 100 μg of MAP-rich tubulin with 100 μM chlorpyrifos, chlorpyrifos oxon, methamidophos, paraoxon, diazinon, diazoxon, monocrotophos, or dichlorvos. Each sample was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue. Five gel slices (at about 30, 50, 150, and 300 kDa, and the top of the separating gel) were removed from the lanes for each of the eight OP samples and from untreated control lanes. These gel slices were subjected to in-gel trypsin digestion. MSMS fragmentation spectra of the tryptic peptides were examined for isopeptide cross-links. Sixteen spectra yielded convincing evidence for isopeptide cross-linked peptides. Ten were from the chlorpyrifos oxon reaction, 1 from dichlorvos, 1 from paraoxon, 1 from diazinon, and 3 from diazoxon. It was concluded that catalysis of protein cross-linking is a general property of organophosphorus pesticides and pesticide metabolites. Data are available via ProteomeXchange with identifier PXD034529.

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, Zn α and Zn β in the active site are five- and six-coordinated, respectively, while both zinc ions are six coordinated with the Zn α -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 Zn α -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 Zn α -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., (© 2022 Wiley-VCH GmbH.)

Acetylcholinesterase (AChE) is reversibly inhibited by α-tocopherol (α-T). Steady state kinetic analysis shows that α-T is a mixed slow-binding inhibitor of type A of human enzyme (K ci  = 0.49 μM; K ui  = 1.6 μM) with a residence time of 2 min on target. Molecular dynamics (MD) simulations support this mechanism, and indicate that α-T first forms multiple non-specific interactions with AChE surface near the gorge entrance, then binds to the peripheral side with alkylene chain slowly sliding down the gorge, inducing no significant conformational change. α-T slightly modulates the progressive inhibition of AChE by the cyclic organophosphorus, cresyl saligenylphosphate, accelerating the fast pseudo-first order process of phosphorylation. A moderate accelerating effect of α-T on phosphorylation by paraoxon was also observed after pre-incubation of AChE in the presence of α-T. This accelerating effect of α-T on ex vivo paraoxon-induced diaphragm muscle weakness was also observed. The effect of α-T on AChE phosphylation was interpreted in light of molecular modeling results. From all results it is clear that α-T does not protect AChE against phosphylation by organophosphorus., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Melanin is a ubiquitous natural pigment found in a diverse array of organisms. Allomelanin is a class of nitrogen-free melanin often found in fungi. Herein, we find artificial allomelanin analogues exhibit high intrinsic microporosity and describe an approach for further increasing and tuning that porosity. Notably, the synthetic method involves an oxidative polymerization of 1,8-DHN in water, negating the need for multiple complex templating steps and avoiding expensive or complex chemical precursors. The well-defined morphologies of these nanomaterials were elucidated by a combination of electron microscopy and scattering methods, yielding to high-resolution 3D reconstruction based on small-angle X-ray scattering (SAXS) results. Synthetic allomelanin nanoparticles exhibit high BET areas, up to 860 m 2 /g, and are capable of ammonia capture up to 17.0 mmol/g at 1 bar. In addition, these nanomaterials can adsorb nerve agent simulants in solution and as a coating on fabrics with high breathability where they prevent breakthrough. We also confirmed that naturally derived fungal melanin can adsorb nerve gas simulants in solution efficiently despite lower porosity than synthetic analogues. Our approach inspires further analysis of yet to be discovered biological materials of this class where melanins with intrinsic microporosity may be linked to evolutionary advantages in relevant organisms and may in turn inspire the design of new high surface area materials.

Enzyme promiscuity is critical to the acquisition of evolutionary plasticity in cells and can be recruited for high-value chemical synthesis or xenobiotic degradation. The molecular determinants of substrate ambiguity are essential to this activity; however, these details remain unknown. Here, we performed the directed evolution of a prolidase to enhance its initially weak paraoxonase activity. The in vitro evolution led to an unexpected 1,000,000-fold switch in substrate selectivity, with a 30-fold increase in paraoxon hydrolysis and 40,000-fold decrease in peptide hydrolysis. Structural and in silico analyses revealed enlarged catalytic cavities and substrate repositioning as responsible for rapid catalytic transitions between distinct chemical reactions., (© 2020 Wiley Periodicals LLC.)

Enzyme engineering for gain of function requires navigating a large combinatorial sequence space efficiently. Typically, many mutations are needed to get significant improvements, while a single "bad" mutation can inactivate the enzyme. To establish high-throughput screening and achieve enhanced resolution between two variants, genetic libraries of the organophosphate hydrolase enzyme paraoxonase 1 (PON1) were rapidly screened via an engineered positive-feedback circuit: a p -nitrophenol (PNP)-specific transcription factor (TF) regulated expression of PON1, which catalyzed paraoxon breakdown and PNP production. Rare active mutant colonies, picked by simple visual fluorescence of a PON1-green fluorescent protein (GFP) fusion, were characterized. In a single screening round, high (library-scale) throughput enabled the discovery of enhanced paraoxon degradation activity in PON1, including structurally unexpected mutations.

Organophosphorus compounds (OP) are highly toxic molecules used as insecticides that inhibit cholinesterase enzymes involved in neuronal transmission. The intensive use of OP for vector control and agriculture has led to environmental pollutions responsible for severe intoxications and putative long-term effects on humans and wild animals. Many in vivo models were studied over the years to assess OP acute toxicity, but the long-term effects are poorly documented. Planarian, a freshwater flatworm having a cholinergic system, has emerged as a new original model for addressing both toxicity and developmental perturbations. We used Schmidtea mediterranea planarians to evaluate long-term effects of paraoxon-ethyl at two sublethal concentrations over three generations. Toxicity, developmental perturbations and disruption of behavior were rapidly observed and higher sensitivity to paraoxon-ethyl of next generations was noticed suggesting that low insecticide doses can induce transgenerational effects. With the view of limiting OP poisoning, SsoPox, an hyperthermostable enzyme issued from the archaea Saccharolobus solfataricus, was used to degrade paraoxon-ethyl prior to planarian exposure. The degradation products, although not lethal to the worms, were found to decrease cholinesterase activities for the last generation of planarians and to induce abnormalities albeit in lower proportion than insecticides.

The single residue mutation of butyrylcholinesterase (BChE G117H ) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in K M and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. In this report, we review recent data showing that decarbamoylation rate constants are independent of the ester leaving group for a series of carbamic acid esters with the same carbamoyl group and that decarbamoylation rate constants decreased by 800-fold when the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-diethyl-. We also review data showing that solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site in N,N-diethylcarbamoyl AChE. The nature of such a conformational change is suggested from X-ray crystal structures of AChE phosphorylated by paraoxon., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of the poison entering the body could be achieved with the help of DNA- or RNA-aptamers, which are able to bind poisons in the bloodstream. The aim of the research was to develop an approach to rational in silico design of aptamers for OPs based on the example of paraoxon. From the published sequence of an aptamer binding organophosphorus pesticides, its three-dimensional model has been constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy have been calculated using MD trajectories and MM-PBSA approach. On the basis of the energy values, two sequences that bind paraoxon most efficiently have been selected. The value of free binding energy of paraoxon with peripheral anionic site of acetylcholinesterase (AChE) has been calculated as well. It has been revealed that the aptamers found bind paraoxon more effectively than AChE. The peculiarities of paraoxon interaction with the aptamers nucleotides have been analyzed. The possibility of improving in silico approach for aptamer selection is discussed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Organophosphorus flame retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 ( Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the p K a of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Brønsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates.

Bacterial biofilms can be programmed to produce living materials with self-healing and evolvable functionalities. However, the wider use of artificial biofilms has been hindered by limitations on processability and functional protein secretion capacity. We describe a highly flexible and tunable living functional materials platform based on the TasA amyloid machinery of the bacterium Bacillus subtilis. We demonstrate that genetically programmable TasA fusion proteins harboring diverse functional proteins or domains can be secreted and can assemble into diverse extracellular nano-architectures with tunable physicochemical properties. Our engineered biofilms have the viscoelastic behaviors of hydrogels and can be precisely fabricated into microstructures having a diversity of three-dimensional (3D) shapes using 3D printing and microencapsulation techniques. Notably, these long-lasting and environmentally responsive fabricated living materials remain alive, self-regenerative, and functional. This new tunable platform offers previously unattainable properties for a variety of living functional materials having potential applications in biomaterials, biotechnology, and biomedicine.

The molecular targets of best known neurotoxic effects associated to acute exposure to organophosphorus compounds (OPs) are serine esterases located in the nervous system, although there are other less known neurotoxic adverse effects associated with chronic exposure to OPs whose toxicity targets are still not identified. In this work we studied sensitivity to the non-neuropathic OP paraoxon and to the neuropathic OP mipafox of phenyl valerate esterases (PVases) in intact and lysed human neuroblastoma SH-SY5Y cells. The main objective was to discriminate different unknown pools of esterases that might be potential targets of chronic effects from those esterases already known and recognized as targets to these acute neurotoxicity effects. Two components of PVases of different sensitivities were discriminated for paraoxon in both intact and lysed cells; while the two components inhibitable by mipafox were found only for intact cells. A completely resistant component to paraoxon of around 30% was found in both intact and lysed cells; while a component of slightly lower amplitude (around 20%) completely resistant to mipafox was also found for both preparations (intact and lysed cells). The comparison of the results between the intact cells and the lysed cells suggests that the plasma membrane could act as a barrier that reduced the bioavailability of mipafox to PVases. This would imply that the discrimination of the different esterases should be made in lysed cells. However, those studies which aim to determine the physiological role of these esterases should be necessarily conducted in intact cultured cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Paraoxonase 1 (PON1) is calcium dependent enzyme involved in many functions in human body. PON1 is synthesized in the liver and secreted to the bloodstream where bounds high-density lipoproteins (HDL). Association of PON1 with HDL increases the enzyme stability and biological activities. PON1 have three different activities: phosphotriesterase, lactonase and arylesterase. Until now there is now commercial available kits to determine these three PON1 activities. Also there is no date about stability of PON1 in serum after storage condition. We have elaborated the optimal conditions for determination of PON1 activities in serum using manual procedure as well as the best storage temperature of human serum for determination of PON1 activities. We have also confirmed that PON1 in serum is associated with HDL. Additionally we have investigated the effect of D-penicillamine, ethylenediaminetetraacetic acid and cadmium chloride on PON1 activities in human serum. D-penicillamine and ethylenediaminetetraacetic acid in therapeutic doses as well as cadmium chloride in toxic doses decrease PON1 activities in human serum when compared to non-treated serum. D-penicillamine as metal chelator inhibits much stronger PON1 activities than ethylenediaminetetraacetic acid., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Organophosphate (OP) poisoning is a major global health issue; while compounds from this group have been used intensively over the last century, an effective antidote is still lacking. Oxime-type acetylcholinesterase (AChE) reactivators are used to reactivate the OP inhibited AChE. Pralidoxime is the only US Food and Drug Administration approved oxime for therapeutic use but its efficacy has been disappointing. Two novel oximes (K378 and K727) were investigated in silico and in vitro and compared with an experimental oxime (kamiloxime; K-27) and pralidoxime. In silico the molecular interactions between AChE and oximes were examined and binding energies were assessed. LogP (predicted log of the octanol/water partition coefficient) was estimated. In vitro the intrinsic ability of the oximes to inhibit AChE (IC 50 ) and their reactivation potency (R 50 ) when used in paraoxon inhibited human RBC-AChE was determined. Molecular docking revealed that K378 and K727 bind to the peripheral site(s) with high binding energies in contrast to the central binding of K-27 and pralidoxime. LogP values indicating that the novel compounds are significantly less hydrophilic than K-27 or pralidoxime. IC 50 of K378 and K727 were comparable (0.9 and 1 µM, respectively) but orders of magnitude lower than comparators. R 50 values revealed their inability to reactivate paraoxon inhibited AChE. It is concluded that the novel oximes K378 and K727 are unlikely to be clinically useful. The in silico and in vitro studies described allow avoidance of unnecessary in vivo animal work and contribute to the reduction of laboratory animal use.

We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly-l-lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26-35 nm). The three copolymer-complexed enzymes exhibited reduced k cat (30-33% reduction), but no significant changes in K m . Inhibitory potency (as reflected by the bimolecular rate constant, k i ) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in k i (16-60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Electrode materials play a key role in the development of electrochemical sensors, particularly enzyme-based biosensors. Here, a novel NiCo 2 S 4 with reticulated hollow spheres assembled from rod-like structures was prepared by a one-pot solvothermal method and its formation mechanism was discussed. Moreover, comparison of NiCo 2 S 4 materials from different experiment conditions as biosensors was investigated by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV), and the best one that was reticulated hollow spheres assembled from rod-like structures NiCo 2 S 4 has been successfully employed as a matrix of AChE immobilization for the special structure, superior conductivity and rich reaction active sites. When using common two kinds of organophosphate pesticides (OPs) as model analyte, the biosensors demonstrated a wide linear range of 1.0×10 -12 -1.0×10 -8 gmL -1 with the detection limit of 4.2×10 -13 gmL -1 for methyl parathion, and 1.0×10 -13 -1.0×10 -10 gmL -1 with the detection limit of 3.5×10 -14 gmL -1 for paraoxon, respectively. The proposed biosensors exhibited many advantages such as acceptable stability and low cost, providing a promising tool for analysis of OPs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A catalytic antibody A17 and its mutants highly efficiently interact with organophosphorus pesticide paraoxon. In this work, we studied the protective properties of antibody A17-K47 in paraoxon poisoning using a mouse model. The optimal paraoxon dose simulating the acute toxic effect of organophosphorus compounds was 550 μg/kg. The pharmacokinetic parameters of A17-K47 antibody were t 1/2distr =7.2±1.4 min, t 1/2el =330±20 min. The antibody did not cause toxic effects when administered at a ten-fold calculated therapeutic dose (610 mg/kg). The drug did not reduce mortality from acute paraoxon poisoning; however, the absence of drug toxicity opens up prospects for its use in symptomatic treatment of chronic paraoxon poisoning.

Assays for organophosphorus pesticides (OPs) with high sensitivity as well as on-site screening have been urgently required to protect ecosystem and prevent disease. Herein, a novel fluorimetric sensing platform was constructed for quantitative detection of OPs via tyrosinase (TYR) enzyme-controlled quenching of gold nanoclusters (AuNCs). One-step green synthetic approach was developed for the synthesis of AuNCs by using chicken egg white (CEW) as template and stabilizer. Initially, TYR can catalyze the oxidation of dopamine to dopaminechrome, which can efficiently quench the fluorescence intensity of AuNCs at 630nm based on dynamic quenching process. However, with the presence of OPs, the activity of TYR was inhibited, resulting in the fluorescence recovery of AuNCs. This proposed fluorescence platform was demonstrated to enable rapid detection for OPs (paraoxon as model) and to provide excellent sensitivity with a detection limit of 0.1ngmL -1 . Significantly, the fluorescence probe was used to prepare paper-based test strips for visual detection of OPs, which validated the excellent potential for real-time and on-site application., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Serum paraoxonase 1 (PON1), an enzyme associated with high - density lipoproteins (HDL) particles, inhibits the oxidation of serum lipoproteins and cell membranes. PON1 activity is lower in patients with atherosclerosis and in inflammatory diseases. The systemic inflammatory response provoked during cardiopulmonary bypass grafting may contribute to the development of postoperative complications. The aim of the present study was to estimate the dynamic changes in paraoxonase 1 (PON1) activity towards paraoxon and phenyl acetate during and after coronary artery surgery., Methods: Twenty six patients with coronary heart disease undergoing coronary artery bypass grafting (CABG) were enrolled into the study. Venous blood samples were obtained preoperatively, after aortic clumping, after the end of operation, at 6, 18, 30 and 48 h after operation. Paraoxonase activity was measured spectrophotometrically in 50 mM glycine/NaOH buffer (pH 10.5) containing 1.0 mM paraoxon, and 1.0 mM CaCl 2 . Arylesterase activity was measured in 20 mM TrisCl buffer (pH 8.0) containing 1 mM phenyl acetate and 1 mM CaCl 2 ., Results: PON1 activity toward paraoxon and phenyl acetate significantly decreased after aorta cross clumping and increased directly after operation. PON1 activity towards paraoxon in preoperative period and PON1 activity towards phenyl acetate in seventh stage of experiment tended to inversely correlate with the occurrence of postoperative complications., Conclusion: The paraoxonase 1 plasma activity is markedly reduced during CABG surgery.

Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70-81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1's lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1's lactonase activity is minimal, whereas the k cat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1's active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar "gating loop" or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates., Competing Interests: The authors declare no competing financial interest.

A new paper-based biosensing approach has been developed for sensitive and rapid detection of acetylcholinesterase (AChE) inhibitors. The biosensing zone of the paper strip is constructed with an inkjet printing method, and the biomolecule AChE is immobilized into two layers of biocompatible sol-gel-derived silica ink with a "sandwich" form. Indoxyl acetate (IDA) is used as a chromogenic substrate, which is colorless and can be catalytically hydrolyzed into blue-colored indigo dipolymer. When the enzymatic activity of AChE is inhibited after incubation with organophosphate pesticides (OPs), there is a decreased hydrolysis of IDA accompanying with a drop in color intensity. Paraoxon and trichlorfon are used as the representative OPs in the assay. Due to the low solubility and high molar absorption coefficient of the IDA dipolymer product, the paper-based strip can form a neat blue sensing zone and shows obviously improved sensitivity with a limit of detection (LOD) of 0.01ngmL -1 paraoxon and 0.04ngmL -1 trichlorfon (S/N=3) and the LODs for visual detection are 0.03ngmL -1 for paraoxon and 0.1ngmL -1 for trichlorfon comparing with the previously reported colorimetric methods. The concentrations of paraoxon in apple juice samples are also detected, and the results are in accord well with these results from high-performance liquid chromatography, showing great potential for on-site detection of OPs in practical application. The developed assay can be used to qualitatively and semiquantitatively estimate with naked eyes and quantitatively assess OPs through image analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The high number of annual fatalities following suicidal poisoning by organophosphorus (OP) pesticides and the recent homicidal use of the chemical warfare nerve agent sarin against civilian population in Syria underlines the continuous threat by these highly toxic agents. The need for an effective treatment of OP poisoning resulted in the implementation of a combination therapy with the muscarinic receptor antagonist atropine and an oxime for the reactivation of OP-inhibited acetylcholinesterase (AChE). Since the invention of the first clinically used oxime pralidoxime (2-PAM) in the 1950s ongoing research attempted to identify more effective oximes. In fact, several thousand oximes were synthesized in the past six decades. These include charged and non-charged compounds, mono- and bispyridinium oximes, asymmetric oximes, oximes with different substitutes and more recently non-oxime reactivators. Multiple in vitro and in vivo studies investigated the potential of oximes to reactivate OP-inhibited AChE and to reverse OP-induced cholinergic signs. Depending on the experimental model, the investigated species and the tested OP largely variable results were obtained by different laboratories. These findings and the inconsistent effectiveness of oximes in the treatment of OP-pesticide poisoned patients led to a continuous discussion on the value of oximes. In order to provide a forward-looking evaluation of the significance of oximes in OP poisoning multiple aspects, including intrinsic toxicity, in vitro reactivation potency, efficacy and pharmacokinetics, as well as the impact of the causative OP have to be considered. The different influencing factors in order to define the benefit and limitations of oximes in OP poisoning will be discussed., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Highly active antiretroviral therapy (HAART), especially protease inhibitors (PIs), commonly used in HIV-infected patients, effectively suppresses a viral replication. However, it is frequently associated with significant side effects, including fat redistribution, lipodystrophy, hyperlipidemia, insulin resistance and diabetes mellitus. Currently, metabolic complications and atherosclerosis resulting from them become the major cause of mortality in HIV-infected patients receiving HAART. Paraoxonase 1 (PON1) is the HDL-bound esterase, which inhibits development of atherosclerosis by decomposing lipid peroxidation products and hydrolyzing homocysteine thiolactone. The aim of this study was to characterize the effects of HIV protease inhibitors on PON1 activity, total plasma homocysteine and protein-bound homocysteine thiolactone as well as lipid profile in rats. The study was performed on seven groups of male Wistar rats: (1) control; (2) and (3) receiving ritonavir (RTV) at doses of 10 and 50 mg/kg, respectively; (4) and (5) receiving atazanavir (ATV) at 10 and 100 mg/kg, respectively; (6) and (7) receiving saquinavir (SQV) at 10 and 50 mg/kg, respectively. All drugs were administered orally for 4 weeks. Compared to control animals, rats receiving PIs had significantly higher concentration of triglycerides and total cholesterol, but the levels of HDL-cholesterol were not different between groups. PON1 activity toward paraoxon was decreased in groups receiving PIs (control: 149 ± 5 U/ml; PIs-treated: RTV at doses 10 mg/kg 133 ± 9.5  U/ml, RTV at doses 50 mg/kg 134 ± 10.8 U/ml, SQV at doses 10 mg/kg 131 ± 9.2 U/ml, ATV at doses 10 mg/kg 132 ± 11.8 U/ml, ATV at doses 100 mg/kg 108 ± 7.8 U/ml). ATV reduced total homocysteine level around 25-28%, whereas other PIs had no effect on its concentration. In contrast, 10-15% increase in protein-bound homocysteine thiolactone was observed in PIs-receiving groups, such as RTV10, RTV50, SQV50, ATV10. In conclusion, dyslipidemia induced by PIs is associated with reduced PON1 activity as well as increased protein homocysteinylation. PON1 deficiency may contribute to increased risk of atherosclerosis in these patients., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to 'sense' a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10(7) variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (∼2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work., (Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

A sensitive amperometric acetylcholinesterase (AChE) biosensor, based on gold nanorods (AuNRs), was developed for the detection of organophosphate pesticide. Compared with Au@Ag heterogeneous NRs, AuNRs exhibited excellent electrocatalytic properties, which can electrocatalytically oxidize thiocholine, the hydrolysate of acetylthiocholine chloride (ATCl) by AChE at +0.55V (vs. SCE). The AChE/AuNRs/GCE biosensor was fabricated on basis of the inhibition of AChE activity by organophosphate pesticide. The biosensor could detect paraoxon in the linear range from 1nM to 5μM and dimethoate in the linear range from 5nM to 1μM, respectively. The detection limits of paraoxon and dimethoate were 0.7nM and 3.9nM, which were lower than the reported AChE biosensor. The proposed biosensor could restore to over 95% of its original current, which demonstrated the good reactivation. Moreover, the biosensor can be applicable to real water sample measurement. Thus, the biosensor exhibited low applied potential, high sensitivity and good stability, providing a promising tool for analysis of pesticides., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Epidemiological evidences indicate close association between inorganic arsenic exposure via drinking water and cardiovascular diseases. While the exact mechanism of this arsenic-mediated increase in cardiovascular risk factors remains enigmatic, epidemiological studies indicate a role for paraoxonase 1 (PON1) in cardiovascular diseases. To investigate the association between inorganic arsenic exposure and cardiovascular diseases, rats were exposed to sodium arsenite (trivalent; 50, 100, and 150 ppm As) and sodium arsenate (pentavalent; 100, 150, and 200 ppm As) in their drinking water for 12 weeks. PON1 activity towards paraoxon (PONase) and phenylacetate (AREase) in plasma, lipoproteins, hepatic, and brain microsomal fractions were determined. Inhibition of PONase and AREase in plasma and HDL characterized the effects of the two arsenicals. While the trivalent arsenite inhibited PONase by 33% (plasma) and 46% (HDL), respectively, the pentavalent arsenate inhibited the enzyme by 41 and 34%, respectively. AREase activity was inhibited by 52 and 48% by arsenite, whereas the inhibition amounted to 72 and 67%, respectively by arsenate. The pattern of inhibition in plasma and HDL indicates that arsenite induced a dose-dependent inhibition of PONase whereas arsenate induced a dose-dependent inhibition of AREase. In the VLDL + LDL, arsenate inhibited PONase and AREase while arsenite inhibited PONase. In the hepatic and brain microsomal fractions, only the PONase enzyme was inhibited by the two arsenicals. The inhibition was more pronounced in the hepatic microsomes where a 70% inhibition was observed at the highest dose of pentavalent arsenic. Microsomal cholesterol was increased by the two arsenicals resulting in increased cholesterol/phospholipid ratios. Our findings indicate that decreased PON1 activity observed in arsenic exposure may be an incipient biochemical event in the cardiovascular effects of arsenic. Modulation of PON1 activity by arsenic may also be mediated through changes in membrane fluidity brought about by changes in the concentration of cholesterol in the microsomes., (© 2014 Wiley Periodicals, Inc.)

The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This work introduced an efficient immobilization of acetylcholinesterase (AChE) onto amino functionalized carbon nanotubes (CNT-NH2), in order to fabricate high sensitive and practical organophosphorus pesticide (OPs) biosensors. Compared with the pristine, -COOH and -OH decorated CNTs, there were larger amount of enzymes adsorbed on the surface of CNT-NH2 with a favorable orientation and the best amperometric response was obtained on the AChE/CNT-NH2/GC electrode. Furthermore, the biosensor modified with CNT-NH2 showed a high affinity to acetylthiocholine chloride (ATCh) and could catalyze the hydrolysis of ATCh with an apparent Michaelis-Menten constant (Km) value of 67.4 µM. Using paraoxon as a model compound, wide linear ranges from 0.2 nM to 1 nM and 1 nM to 30 nM, and a low detection limit of 0.08 nM were obtained with satisfactory reproducibility and stability. Moreover, the biosensor had also been successfully employed for the determination of low concentrations of pesticides in real vegetable samples. This method could be extended to other functionalized nano-materials for their application in constructing biosensors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.

Aims: Organophosphorus pesticides are widely used in agriculture. Accordingly, decontamination of these pesticides and their residual in environment is an important aim of researchers. One of the best approaches is enzymatic detoxification of these compounds with organophosphorus hydrolase (OPH). The immobilization of OPH on environmentally friendly supports is of great importance for developing stabilized enzymes for degradation of organophosphorus compounds., Methods and Results: In this study, Bacillus subtilis spores were applied as a new matrix for immobilizing OPH for the first time; this enzyme was covalently bound to the spores by using EDC-NHS as coupling reagents and the immobilization was confirmed by enzymatic activity, Western blot, flow cytometry and fluorescence microscopic analysis. The immobilization yield was about 55% and the immobilized OPH hydrolysed paraoxon, an organophosphate substrate, without significant loss of activity was six times. The spores with immobilized OPH on their surface were successfully characterized using FT-IR analysis and SEM imaging. Thermal and pH stability was improved by immobilization of OPH on the spore surface., Conclusions: Owing to safety, environmentally friendly and low cost of spores, these spores can be employed in biosensors for monitoring and biodegradation of organophosphate contaminants in the environment and detoxification processes in bioreactors with high reusability without decrease in the activity., Significance and Impact of the Study: We believe that the spore, an environmentally friendly matrix, can be used for covalent immobilization of OPH efficiently and can be applied for detoxification of organophosphorus compounds under adverse environmental conditions., (© 2015 The Society for Applied Microbiology.)

Introduction: Paraoxonase 1 (PON1) is an antioxidative enzyme manly associated with high density lipoproteins (HDL) in the peripheral blood. The aim of this study was to determine the PON1 paraoxonase and arylesterase activities in patients with chronic obstructive pulmonary disease (COPD). We also aimed to determine the concentration of reduced thiol groups as a marker of protein oxidation., Materials and Methods: The study included 105 patients with stable COPD and 44 healthy controls. PON1 activities and thiols concentration were assayed in sera by spectrophotometry., Results: PON1 basal (POX) and salt-stimulated paraoxonase activity (POX1) as well as arylesterase activity (ARE) were significantly reduced in COPD patients. In addition, concentration of reduced thiol groups was significantly decreased in COPD group. PON1 activities were similar in patients with different disease severity (GOLD stages). However, a significant reduction in POX, POX1 and ARE was observed already in GOLD II stage when compared to controls. POX and POX1 showed modest while ARE yielded very good power for discrimination between healthy subjects and COPD patients. Univariate and multivariate logistic regression analysis indicated that ARE is a good COPD predictor., Conclusion: Reduction of PON1 activity observed in COPD patients could be partly caused by oxidative environment. Lower concentrations of reduced thiol groups in COPD patients suggest that a decrease in PON1 activity could reflect oxidative changes of enzyme free cysteine residues. Furthermore, decreased PON1 arylesterase activity might indicate a down-regulation of PON1 concentration. Our results suggest that ARE could be considered as potential biomarker for COPD diagnosis.

Phosphatases for organophosphate degradation and carbohydrate-binding domains (CBMs) have potential biotechnological applications. As a proof-of-concept, a soluble chimeric protein that combines acid phosphatase (AppA) from Escherichia coli and a CBM from Xanthomonas axonopodis pv. citri (AppA-CBM) was produced in E.coli. AppACBM adsorbed in microcrystalline cellulose Avicel PH101 catalyzed the hydrolysis of p-nitrophenyl phosphate (PNPP). The binding to microcrystalline cellulose displayed saturation behavior with an apparent binding constant (Kb) of 22 ± 5 mg and a maximum binding (Bmax) of 1.500 ± 0.001 enzyme units. Binding was highest at pH 2.5 and decreased above pH 6.5, as previously observed for family 2 CBMs. The Km values for PNPP of AppA-CBM and native AppA were identical (2.7 mM). To demonstrate that this strategy for protein engineering has practical applications and is largely functional, even for phosphatases exhibiting diverse folds, a chimeric protein combining human paraoxonase 1 (hPON1) and the CBM was produced. Both PON1-CBM and hPON1 had identical K m values for paraoxon (1.3 mM). Additionally, hPON1 bound to microcrystalline cellulose with a Kb of 27 ± 3 mg, the same as that observed for AppA-CBM. These data show that the phosphatase domains are as functional in both of the chimeric proteins as they are in the native enzymes and that the CBM domain maintains the same cellulose affinity. Therefore, the engineering of chimeric proteins combining domains of phosphatases and CBMs is fully feasible, resulting in chimeric enzymes that exhibit potential for OP detoxification.

In this study, paraoxonase 1 (PON1; EC 3.1.8.1) was purified from bull semen, and some characteristics of the enzyme were investigated. In vitro inhibition effect of some heavy metals, including Cu(2+), Mn(2+), Cd(2+), Zn(2+), Ni(2+), and Pb(2+), on the activity of the purified enzyme was also investigated. The purification of bull semen PON1 procedure was composed of two steps: ammonium sulfate precipitation and Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The enzyme, having a specific activity of 288 EU/mg proteins, was purified 22.67-fold with a yield of 89 %. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme showed the presence of a single band with an apparent MW of 66 kDa. The V max and K M values for the paraoxon substrate were determined as 100 EU and 8.0 × 10(-5) M, respectively. The inhibitory effects of different heavy metals on PON1 activity were determined by using the paraoxon as a substrate. The results showed that all the metals, except for Cd(2+), inhibited the PON1 enzyme activity in a concentration-dependent fashion. IC50 values of Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) were found as 2.59 × 10(-3), 1.17 × 10(-3), 42.74 × 10(-3), 99.10 × 10(-3), 48.80 × 10(-3) mM, respectively. Conversely, Cd(2+) increased the bull semen PON1 enzyme activity. The present study has demonstrated that Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) are serious toxic metals, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.

Objective: To investigate early markers of atherosclerosis in patients with nasal polyposis (NP) through measurements of carotid artery intima-media thickness (CIMT), flow-mediated vasodilatation (FMD) of the brachial artery and serum paraoxonase-1 (PON-1) activity., Methods: Forty-five patients with NP were included in the study group and 45 healthy individuals in the control group. The diagnosis of patients with NP was predicated on anterior rhinoscopy, endoscopic nasal examination and coronal paranasal sinus computed tomography (CT). Measurements of CIMT and FMD of the brachial artery were performed by high-resolution ultrasonography. Serum PON-1 activity was evaluated by measuring the rate of paraoxon hydrolysis., Results: Mean CIMT values were found to be increased in the NP group compared to the control group. However, mean FMD % values and serum PON-1 activity were significantly lower in the NP group compared to the control group. Moreover; the endoscopic polyps' scores and paranasal sinus CT scores were positively correlated with CIMT and negatively correlated with FMD % values and PON-1 activity. Disease duration also was positively correlated with CIMT and negatively correlated with FMD % values., Conclusion: Impaired FMD, increased CIMT and decreased serum PON-1 activity may be considered to be risk factors for accelerated atherosclerosis in patients with NP who may have subclinical atherosclerosis and be at risk for cardiovascular events in the future., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Decontamination of soils with complex pollution using natural strains of microorganisms is a matter of great importance. Here we report that oil-oxidizing bacteria Rhodococcus erythropolis AC-1514D and Rhodococcus ruber AC-1513D can degrade various organophosphorous pesticides (OP). Cell-mediated degradation of five different OP is apparently associated with the presence of N-acylhomoserine lactonase, which is pronouncedly similar (46-50 %) to the well-known enzyme organophosphate hydrolase (OPH), a hydrolysis catalyst for a wide variety of organophosphorous compounds. Additionally, we demonstrated the high lactonase activity of hexahistidine-tagged organophosphate hydrolase (His6-OPH) with respect to various N-acylhomoserine lactones, and we determined the catalytic constants of His6-OPH towards these compounds. These experimental data and theoretical analysis confirmed the hypothesis about the evolutionary proximity of OPH and lactonases. Using Rhodococcus cells, we carried out effective simultaneous biodegradation of pesticide paraoxon (88 mg/kg) and oil hydrocarbon hexadecane (6.3 g/kg) in the soil. Furthermore, the discovered high lactonase activity of His6-OPH offers new possibilities for developing an efficient strategy of combating resistant populations of Gram-negative bacterial cells.

Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k cat, K m, and k cat/K m). Continuous spectroscopic assays are frequently only possible by using synthetic (i.e., nonbiological) substrates that possess a suitable chromophoric marker (e.g., nitrophenol). Isothermal titration calorimetry, in contrast, affords a rapid assay independent of the chromophoric properties of the substrate-the heat associated with the hydrolytic reaction can be directly related to catalytic properties. Here, we demonstrate the efficiency of the method on several selected examples of this family of enzymes and show that, in general, the catalytic parameters obtained by isothermal titration calorimetry are in good agreement with those obtained from spectroscopic assays.

Organophosphates (OPs) widely exist in ecosystem as toxic substances, for which sensitive and rapid analytical methods are highly requested. In the present work, by using N-terminal of ice nucleation protein (INP) as anchoring motif, a genetically engineered Escherichia coli (E. coli) strain surface displayed mutant organophosphorus hydrolase (OPH) (S5) with improved enzyme activity was successfully constructed. The surface location of INP-OPH fusion was confirmed by SDS-PAGE analysis and enzyme activity assays. The OPH-displayed bacteria facilitate the hydrolysis of p-nitrophenol (PNP) substituted organophosphates to generate PNP, which can be detected spectrometrically at 410 nm. Over 90% of the recombinant protein present on the surface of microbes demonstrated enhanced enzyme activity and long-term stability. The OPH activity of whole cells was 2.16 U/OD₆₀₀ using paraoxon as its substrate, which is the highest value reported so far. The optimal temperature for OPH activity was around 55 °C, and suspended cultures retained almost 100% of its activity over a period of one month at room temperature, exhibiting the better stability than free OPH. The recombinant E. coli strain could be employed as a whole-cell biocatalyst for detecting PNP substituted OPs at wider ranges and lower detection limits. Specifically, the linear ranges of the calibration curves were 0.5-150 μM paraoxon, 1-200 μM parathion and 2.5-200 μM methyl parathion, and limits of detection were 0.2 μM, 0.4 μM and 1 μM for paraoxon, parathion and methyl parathion, respectively (S/N=3). These results indicate that the engineered OPH strain is a promising multifunctional bacterium that could be used for further large-scale industrial and environmental applications., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The organophosphorous hydrolase (PTE) from Brevundimonas diminuta is capable of degrading extremely toxic organophosphorous compounds with a high catalytic turnover and broad substrate specificity. Although the natural substrate for PTE is unknown, its loop remodeling (loop 7-2/H254R) led to the emergence of a homoserine lactonase (HSL) activity that is undetectable in PTE (kcat/km values of up to 2 × 10(4)), with only a minor decrease in PTE paraoxonase activity. In this study, homology modeling and molecular dynamics simulations have been undertaken seeking to explain the reason for the substrate specificity for the wild-type and the loop 7-2/H254R variant. The cavity volume estimated results showed that the active pocket of the variant was almost two fold larger than that of the wild-type (WT) enzyme. pKa calculations for the enzyme (the WT and the variant) showed a significant pKa shift from WT standard values (ΔpKa = 3.5 units) for the His254 residue (in the Arg254 variant). Molecular dynamics simulations indicated that the displacement of loops 6 and 7 over the active site in loop 7-2/H254R variant is useful for N-acyl-L-homoserine lactone (C4-HSL) with a large aliphatic chain to site in the channels easily. Thence the expanding of the active pocket is beneficial to C4-HSL binding and has a little effect on paraoxon binding. Our results provide a new theoretical contribution of loop remodeling to the rapid divergence of new enzyme functions.

Neurotoxic organophosphorus compounds (OPs), which are used as pesticides and chemical warfare agents lead to more than 700,000 intoxications worldwide every year. The main target of OPs is the inhibition of acetylcholinesterase (AChE), an enzyme necessary for the control of the neurotransmitter acetylcholine (ACh). The control of ACh function is performed by its hydrolysis with AChE, a process that can be completely interrupted by inhibition of the enzyme by phosphylation with OPs. Compounds used for reactivation of the phosphylated AChE are cationic oximes, which usually possess low membrane and hematoencephalic barrier permeation. Neutral oximes possess a better capacity for hematoencephalic barrier permeation. NMR spectroscopy is a very confident method for monitoring the inhibition and reactivation of enzymes, different from the Ellman test, which is the common method for evaluation of inhibition and reactivation of AChE. In this work (1)H NMR was used to test the effect of neutral oximes on inhibition of AChE and reactivation of AChE inhibited with ethyl-paraoxon. The results confirmed that NMR is a very efficient method for monitoring the action of AChE, showing that neutral oximes, which display a significant AChE inhibition activity, are potential drugs for Alzheimer disease. The NMR method showed that a neutral oxime, previously indicated by the Ellman test as better in vitro reactivator of AChE inhibited with paraoxon than pralidoxime (2-PAM), was much less efficient than 2-PAM, confirming that NMR is a better method than the Ellman test., (Copyright © 2013. Published by Elsevier Ltd.)

Serum paraoxonase 1 (PON1) was purified from bovine serum using hydrophobic interaction chromotography on Sepharose 4B-coupled l-tyrosine 1-naphthylamine gel, and monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Paraoxonase enzyme was immobilized using different ratios of glutaraldehyde and the maximum activity was observed with 7% glutaraldehyde. The effects of inhibition by Mn(+2), Co(+2) and Cu(+2) heavy metals on the immobilized and free enzyme activities were studied. At the optimum pH and temperature, the K(m) and V(max) kinetic values for bovine serum paraoxonase and immobilized paraoxonase towards paraoxon substrate were determined as 0.296 × 10(-3) M & 37.04 EU vs. 0.727-10(-3) M & 36.36 EU, respectively.

Nanocapsules of organophosphorous hydrolase with enhanced enzyme activity and stability are prepared via in situ polymerization, providing a novel class of nanoparticles for the decontamination and detoxification of organophosphates such as chemical warfare agents and pesticides. Using the nanocapsules as building blocks, bioactive nanocomposites are also fabricated, enabling their use for organophosphate protection., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Takayasu's arteritis (TA) is a chronic inflammation that affects the large vessels; however, its etiology is still unknown. Human serum paraoxonase hydrolyzes oxidized lipids into low-density lipoproteins and could therefore be associated with the prevalence of inflammation processes. Therefore, the purpose of study was to elucidate the influence of PON1 gene polymorphisms and plasma PON1 activities in Takayasu's arteritis disease., Methods: Fifty-four patients with TA and 173 clinically healthy Mexicans were studied. The PON1 polymorphism was determined by the TaqMan PCR method. PON1 activity was assessed spectrophotometrically by paraoxon (p-nitrophenylphosphate) hydrolysis., Results: In TA patients, the frequency of PON1(192R) allele (51% vs. 39%, P=0.043, OR=1.60, 95% CI=1.03-2.47), PON1(55M) allele (21% vs. 6.6%, P=0.0001, OR=3.80, 95% CI=2.03-7.10), and PON1(-108T) (60.1% vs. 46%, P=0.011, OR 1.79 (95% CI=1.15-2.79) were significantly higher than in healthy controls. PON1 activity was significantly lower for PON in TA vs. controls (136.14 vs. 322.79 μmol min(-1) ml(-1), P=0.001, showing a decreasing activity in all genotypes in TA patients with respect to the control subjects., Conclusions: These results show significantly lower PON1 activity associated with HDL-C in TA patients, this activity could be depending on PON1 genotypes; showing that QR/LM/CT has the lowest hydrolytic activity toward paraoxon meanwhile, PON1(192,55,-108) genetic variations are related with reduced PON1 activities, these could be factors contributing to the development of TA disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In an effort to discover novel catalytic bioscavengers of organophosphorus (OP) nerve agents, cell lysates from a diverse set of bacterial strains were screened for their capacity to hydrolyze the OP nerve agents VX, VR, and soman (GD). The library of bacterial strains was identified using both random and rational approaches. Specifically, two representative strains from eight categories of extremophiles were chosen at random. For the rational approach, the protein sequence of organophosphorus hydrolase (OPH) from Brevundimonas diminuta was searched against a non-redundant protein database using the Basic Local Alignment Search Tool to find regions of local similarity between sequences. Over 15 protein sequences with significant sequence similarity to OPH were identified from a variety of bacterial strains. Some of these matches were based on predicted protein structures derived from bacterial genome sequences rather than from bona fide proteins isolated from bacteria. Of the 25 strains selected for nerve agent testing, three bacterial strains had measurable levels of OP hydrolase activity. These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had detectable hydrolysis of VR; Haloarcula sp. had appreciable hydrolysis of VX and VR, whereas lysates from M. aurantiaca had detectable hydrolysis of VR and GD., (Published by Elsevier Ireland Ltd.)

Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus acidocaldarius lactonase, showing low but significant and extremely thermostable paraoxonase activity. This enzyme, that we have named SacPox, is a member of the new described family of phosphotriesterase-like lactonases (PLLs). In this family the binuclear metal centre, which is involved in the catalytic machinery, has been poorly studied up to now. In this work we describe the expression of the protein in presence of different metals showing Mn(2+) to support the higher activity. The enzyme has been over-expressed, purified and characterized as a Mn(2+)-containing enzyme by inductive plasma coupled mass spectrometry (ICP-MS), showing also surprising kinetic differences in comparison with the cadmium-containing enzyme. The Mn(2+) containing enzyme was about 30-fold more efficient with paraoxon as substrate and more stable than the Cd(2+) counterpart, even though the Mn(2+) affinity for the binuclear metal centre is apparently lower. These results increase our knowledge of the biochemical characteristics of SacPox mainly with regard to the metal-ions modulation of function., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Variants of human paraoxonase 1 (PON1) are being developed as catalytic bioscavengers for the organophosphorus chemical warfare agents (OP). It is preferable that the new PON1 variants have broad spectrum hydrolase activities to hydrolyze both G- and V-class OPs. H115W PON1 has shown improvements over wild type PON1 in its capacity to hydrolyze some OP compounds. We improved upon these activities either by substituting a tryptophan (F347W) near the putative active site residues for enhanced substrate binding or by reducing a bulky group (Y71A) at the periphery of the putative enzyme active site. When compared to H115W alone, we found that H115W/Y71A and H115W/F347W maintained VX catalytic efficiency but showed mixed results for the capacity to hydrolyze paraoxon. Testing our double mutants against racemic sarin, we observed reduced values of K(M) for H115W/F347W that modestly improved catalytic efficiency over wild type and H115W. Contrary to previous reports, we show that H115W can hydrolyze soman, and the double mutant H115W/Y71A is nearly 4-fold more efficient than H115W for paraoxon hydrolysis. We also observed modest stereoselectivity for hydrolysis of the P(-) stereoisomer of tabun by H115W/F347W. These data demonstrate enhancements made in PON1 for the purpose of developing an improved catalytic bioscavenger to protect cholinesterase against chemical warfare agents., (Published by Elsevier Ireland Ltd.)