Organophosphorus Pesticides Promote Protein Cross-Linking.

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.

Density functional theory explorations of parathion and paraoxon hydrolysis as a function of the underlying alkaline environment.

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.

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.

Allomelanin: A Biopolymer of Intrinsic Microporosity.

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 m2/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.

Repurposing a bacterial prolidase for organophosphorus hydrolysis: Reshaped catalytic cavity switches substrate selectivity.

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.

Smart Microbial Cells Couple Catalysis and Sensing to Provide High-Throughput Selection of an Organophosphate Hydrolase.

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.

Enzymatic decontamination of paraoxon-ethyl limits long-term effects in planarians.

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.

Engineering Dynamic Surface Peptide Networks on ButyrylcholinesteraseG117H for Enhanced Organophosphosphorus Anticholinesterase Catalysis.

The single residue mutation of butyrylcholinesterase (BChEG117H) 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 KM 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.

Rate-limiting step in the decarbamoylation of acetylcholinesterases with large carbamoyl groups.

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.

Rational in silico design of aptamers for organophosphates based on the example of paraoxon.

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.

Transition State Analysis of the Reaction Catalyzed by the Phosphotriesterase from Sphingobium sp. TCM1.

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 Ka 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.

Programmable and printable Bacillus subtilis biofilms as engineered living materials.

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.

Hydrolyzing activities of phenyl valerate sensitive to organophosphorus compounds paraoxon and mipafox in human neuroblastoma SH-SY5Y cells.

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.

The effect of divalent metal chelators and cadmium on serum phosphotriesterase, lactonase and arylesterase activities of paraoxonase 1.

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.

In silico and in vitro evaluation of two novel oximes (K378 and K727) in comparison to K-27 and pralidoxime against paraoxon-ethyl intoxication.

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 (IC50) and their reactivation potency (R50) 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. IC50 of K378 and K727 were comparable (0.9 and 1 µM, respectively) but orders of magnitude lower than comparators. R50 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.

Polyionic complexes of butyrylcholinesterase and poly-l-lysine-g-poly(ethylene glycol): Comparative kinetics of catalysis and inhibition and in vitro inactivation by proteases and heat.

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 kcat (30-33% reduction), but no significant changes in Km. Inhibitory potency (as reflected by the bimolecular rate constant, ki) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in ki (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.

A Study of the Protective Properties of an Antibody-Based Antidote Metabolizing Organophosphorus Pesticide Paraoxon.

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 t1/2distr =7.2±1.4 min, t1/2el =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.

Dynamic changes of paraoxonase 1 activity towards paroxon and phenyl acetate during coronary artery surgery.

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 CaCl2. Arylesterase activity was measured in 20 mM TrisCl buffer (pH 8.0) containing 1 mM phenyl acetate and 1 mM CaCl2. 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.

Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1.

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 ß-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 kcat 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.

Sensitive inkjet printing paper-based colormetric strips for acetylcholinesterase inhibitors with indoxyl acetate substrate.

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.