Molecular binding of different classes of organophosphates to methyl parathion hydrolase from Ochrobactrum species.

Methyl parathion hydrolase (MPH) is an enzyme of the metallo-ß-lactamase superfamily, which hydrolyses a wide range of organophosphates (OPs). Recently, MPH has attracted attention as a promising enzymatic bioremediator. The crystal structure of MPH enzyme shows a dimeric form, with each subunit containing a binuclear metal ion center. MPH also demonstrates metal ion-dependent selectivity patterns. The origins of these patterns remain unclear but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. We aimed to investigate and compare the binding of different OP pesticides to MPH with cobalt(II) metal ions. In this study, MPH was modeled from Ochrobactrum sp. with different OP pesticides bound, including methyl paraoxon and dichlorvos and profenofos. The docked structures for each substrate optimized by DFT calculation were selected and subjected to atomistic molecular dynamics simulations for 500 ns. It was found that alpha metal ions did not coordinate with all the pesticides. Rather, the pesticides coordinated with less buried beta metal ions. It was also observed that the coordination of beta metal ions was perturbed to accommodate the pesticides. The binding free energy calculations and structure-based pharmacophore model revealed that all the three substrates could bind well at the active site. However, profenofos exhibit a stronger binding affinity to MPH in comparison to the other two substrates. Therefore, our findings provide molecular insight on the binding of different OP pesticides which could help us design the enzyme for OP pesticides degradation.

On-site screening method for bioavailability assessment of the organophosphorus pesticide, methyl parathion, and its primary metabolite in soils by paper strip biosensor.

An important public concern worldwide is soil pollution caused by organophosphorus pesticides and their primary metabolites. To protect the public's health, screening these pollutants on-site and determining their soil bioavailability is important, but doing so is still challenging. This work improved the already-existing organophosphorus pesticide hydrolase (mpd) and transcriptional activator (pobR), and it first designed and constructed a novel biosensor (Escherichia coli BL21/pNP-LacZ) that can precisely detect methyl parathion (MP) and its primary metabolite p-nitrophenol with low background value. To create a paper strip biosensor, E. coli BL21/pNP-LacZ was fixed to filter paper using bio-gel alginate and sensitizer polymyxin B. According to the calibrations of the paper strip biosensor for soil extracts and standard curve, the color intensity of the paper strip biosensor collected by the mobile app may be used to compute the concentration of MP and p-nitrophenol. This method's detection limits were 5.41 µg/kg for p-nitrophenol and 9.57 µg/kg for MP. The detection of p-nitrophenol and MP in laboratory and field soil samples confirmed this procedure. Paper strip biosensor on-site allows for the semi-quantitative measurement of p-nitrophenol and MP levels in soils in a simple, inexpensive, and portable method.

Biological responses to imazapic and methyl parathion pesticides in bioinspired lipid membranes and Tilapia fish.

Pesticide misuse has well-documented detrimental effects on ecosystems, with Nile tilapia (Oreochromis niloticus) being particularly vulnerable. The current study focuses on the impact of widely used sugarcane crop pesticides, Imazapic (IMZ) and Methyl Parathion (MP), on tilapia gill tissues and their lipid membranes. This investigation was motivated by the specific role of the lipid membrane in transport regulation. Bioinspired cell membrane models, including Langmuir monolayers and liposomes (LUVs and GUVs), were utilized to explore the interaction of IMZ and MP. The results revealed electrostatic interactions between IMZ and MP and the polar head groups of lipids, inducing morphological alterations in the lipid bilayer. Tilapia gill tissue exposed to the pesticides exhibited hypertrophic increases in primary and secondary lamellae, total lamellar fusion, vasodilation, and lifting of the secondary lamellar epithelium. These alterations can lead to compromised oxygen absorption by fish and subsequent mortality. This study not only highlights the harmful effects of the pesticides IMZ and MP, but also emphasizes the crucial role of water quality in ecosystem well-being, even at minimal pesticide concentrations. Understanding these impacts can better inform management practices to safeguard aquatic organisms and preserve ecosystem health in pesticide-affected environments.

The adaptive landscape of a metallo-enzyme is shaped by environment-dependent epistasis.

Enzymes can evolve new catalytic activity when environmental changes present them with novel substrates. Despite this seemingly straightforward relationship, factors other than the direct catalytic target can also impact adaptation. Here, we characterize the catalytic activity of a recently evolved bacterial methyl-parathion hydrolase for all possible combinations of the five functionally relevant mutations under eight different laboratory conditions (in which an alternative divalent metal is supplemented). The resultant adaptive landscapes across this historical evolutionary transition vary in terms of both the number of "fitness peaks" as well as the genotype(s) at which they are found as a result of genotype-by-environment interactions and environment-dependent epistasis. This suggests that adaptive landscapes may be fluid and molecular adaptation is highly contingent not only on obvious factors (such as catalytic targets), but also on less obvious secondary environmental factors that can direct it towards distinct outcomes.

Higher-order epistasis shapes the fitness landscape of a xenobiotic-degrading enzyme.

Characterizing the adaptive landscapes that encompass the emergence of novel enzyme functions can provide molecular insights into both enzymatic and evolutionary mechanisms. Here, we combine ancestral protein reconstruction with biochemical, structural and mutational analyses to characterize the functional evolution of methyl-parathion hydrolase (MPH), an organophosphate-degrading enzyme. We identify five mutations that are necessary and sufficient for the evolution of MPH from an ancestral dihydrocoumarin hydrolase. In-depth analyses of the adaptive landscapes encompassing this evolutionary transition revealed that the mutations form a complex interaction network, defined in part by higher-order epistasis, that constrained the adaptive pathways available. By also characterizing the adaptive landscapes in terms of their functional activities towards three additional organophosphate substrates, we reveal that subtle differences in the polarity of the substrate substituents drastically alter the network of epistatic interactions. Our work suggests that the mutations function collectively to enable substrate recognition via subtle structural repositioning.

Flavin-Based Fluorescent Protein EcFbFP Auto-Guided Surface Display of Methyl Parathion Hydrolase in Escherichia coli.

Methyl parathion hydrolase (MPH) plays an important role in degrading a range of organophosphorus compounds. In order to display MPH on the cell surface of Escherichia coli strain RosettaBlue™, the Flavin-based fluorescent protein EcFbFP was severed as an auto-anchoring matrix. With net negative charges of EcFbFP supplying the driving forces, fusion protein MPH-EcFbFP through a two-step auto-surface display process was finally verified by (a) inner membrane translocation and (b) anchoring at outer membrane. Cells with surface-displayed MPH obtained activity of 0.12 U/OD600 against substrate methyl parathion. MPH when fused with engineered EcFbFP containing 20 net negative charges exhibited fivefold higher anchoring efficiency and tenfold higher enzymatic catalytic activity of 1.10 U/OD600. The above result showed that MPH was successfully displayed on cell surface and can be used for biodegradation of methyl parathion.

Role of interfacial reactions in biodegradation: A case study in a montmorillonite, Pseudomonas sp. Z1 and methyl parathion ternary system.

Organophosphate pesticides are currently the most commonly used pesticides, but the mechanisms of biodegradation of these compounds are often unknown. In this study, we constructed a ternary biodegradation system containing methyl parathion (MP), a bacterial strain of Pseudomonas sp. Z1 with capability of degrading MP and montmorillonite, which is a common clay mineral in soils. The role of interfacial reactions between montmorillonite and the MP degrader on the biodegradation of MP was investigated by batch adsorption as well as through semi-permeable membrane experiments. The contact between degrader and montmorillonite in biodegradation was also dynamically examined using in situ attenuated total reflectance Fourier transform infrared spectroscopy. The metabolic activity of the degrading bacteria was also assessed using an isothermal microcalorimetric technique. The results indicate that sorption of bacterial cells onto montmorillonite enhances the metabolic activity of the bacteria and hence the biodegradation of MP by the bacteria, and that an amide group on a bacterial surface protein is responsible for the bacterial adhesion onto the montmorillonite. This stimulated effect ceased when the bacteria were physically separated from the surface of the clay by a membrane, demonstrating the importance of sorption of both the bacteria and the MP in the biodegradation process.

Molecular cloning and characterization of a methyl parathion hydrolase from an organophosphorus-degrading bacterium, Serratia marcescens MEW06.

An organophosphorus-degrading bacterium MEW06, which exhibited excellent biodegradation capabilities towards 50 mg/L of methyl parathion (MP), paraoxon and dimethoate, was isolated from Sand Lake (Wuhan, China) and identified as Serratia marcescens subsp. marcescens based on physiological-biochemical characteristics and a 16S rDNA sequence-based phylogenetic tree. MEW06 genome contains a 31.09-kDa methyl parathion hydrolase (MPH) (MPHGM004539) that was 54.9% similar to Pseudomonas sp. WBC-3's MPH. RT-qPCR revealed that mphGM004539 gene expression was significant up-regulated when co-cultured with MP. mphGM004539 without signal peptide (mphGM004539Δsp) was successful cloned and expressed in Escherichia coli BL21 (DE3). Optimized specific enzyme activity of MPHGM004539ΔSP was 5.26 U/mg under 35°C and pH 11.0 conditions when MP as the substrate. Additionally, Co2+, Cd2+and Fe2+ increased the enzyme activity level. MP could be degraded by MPHGM004539ΔSP into p-nitrophenol probably by hydrolyzing the P-O ester bond. Virulence of MP towards Drosophila melanogaster W1118 was reduced by MEW06 or MPHGM004539ΔSP biodegradation. This is the first cloning and characterization of MPH from the organophosphorus-degrading bacterium S. marcescens. MEW06 and its MPH have potential roles in the bioremediation of organophosphorus pesticide-contaminated eco-systems.

Dual-opposite multi-walled carbon nanotube modified carbon fiber microelectrode for microfluidic chip-capillary electrophoresis determination of methyl parathion metabolites in human urine.

Methyl parathion (MP) is a highly toxic organophosphate and its exposure may lead to substantial adverse effects to human health. The existence of 4-nitrophenol (4-NP) in the form of free phenol, glucuronide (4-NP-G) or as a sulfate ester (4-NP-S) can be used as biomarkers to assess the duration and extent of MP exposure. In this work, a MC-CE device incorporating post-CE amperometric detection using multi-walled carbon nanotubes (MWNTs) modified carbon fiber microelectrode (CFME) was fabricated and assessed for simultaneous determination of 4-NP, 4-NP-G, and 4-NP-S in human urine. The detection sensitivity and stability was greatly enhanced by the modification of MWNTs. The capability of the MC-CE device with dual MWNTs modified CFME for detecting impurity was assessed and reliability established by high recoveries from 95 to 97% for spiked MP biomarkers. The method developed is shown to provide a simple, sensitive, and reliable means for monitoring 4-NP, 4-NP-G, and 4-NP-S in human urine.

Enhancing methyl parathion degradation by the immobilization of Burkholderia sp. isolated from agricultural soils.

Organophosphate pesticides are of great interest for research because they are currently the most commonly used pesticides. In this study, a bacterial strain capable of completely degrading methyl parathion (MP) was isolated from agricultural soils in central Mexico. This strain was designated strain S5-2 and was identified as Burkholderia cenocepacia. To increase degradation yields, cells were immobilized on three different supports: powdered zeolite and Opuntia sp. and Agave sp. fibers. The results indicated a significant increase in MP hydrolysis and p-nitrophenol (PNP) degradation with immobilized cells compared to free cell cultures. Furthermore, immobilized cells were capable of withstanding and degrading higher concentrations of PNP compared to cell suspension cultures. The cell viability in the free cell cultures, as well as PNP degradation, was affected at concentrations greater than 25 mg/L. In contrast, cells immobilized on Opuntia sp. and Agave sp. fibers completely degraded PNP at concentrations of 100 mg/L. To verify that MP solution toxicity was decreased by B. cenocepacia strain S5-2 via pesticide degradation, we measured the acetylcholinesterase activity, both before and after treatment with bacteria. The results demonstrate that the activity of acetylcholinesterase was unaffected after MP degradation by bacteria.

Characterization of methyl parathion degradation by a Burkholderia zhejiangensis strain, CEIB S4-3, isolated from agricultural soils.

Through the use of an enrichment technique, we isolated from the agricultural soils of Morelos in central México a strain of Burkholderia zhejiangensis identified as CEIB S4-3, it's could use the pesticide methyl parathion (MP) as the only source of carbon and degrade completely p-nitrophenol (PNP). For more efficient MP and PNP degradation by the CEIB S4-3 strain, the absence of an extra carbon source, a large inoculum and an MP concentration up to 50 mg/l are required. Sequence and annotation analysis of the draft genome, showed presence of mpd functional gene, which was expressed and its activity on the MP was confirmed. Additionally, the genes coding for enzymes in the benzoquinone pathway (conducted by Gram-negative bacteria) and the benzenotriol pathway (conducted by Gram-positive bacteria) were found, which was corroborated by identification of intermediary metabolites by HPLC. Thus, we propose that B. zhejiangensis CEIB S4-3 uses both degradation pathways.

An optical microplate biosensor for the detection of methyl parathion pesticide using a biohybrid of Sphingomonas sp. cells-silica nanoparticles.

The previously developed Sphingomonas sp. based optical microplate biosensor for methyl parathion (MP) was good as it detected multiple samples but had poor stability and low sensitivity. The present study aims to overcome these limitations. Silica nanoparticles (Si NP) were thus functionalized with polyethyleneimine (PEI) and the functionalized silica nanoparticles (fSi NP) were then integrated with Sphingomonas sp. cells. The process was optimized for hydrolysis of MP into p-nitrophenol (PNP). Integration of fSi NP with cells was confirmed by FT-IR analysis. Biohybrid of Sphingomonas sp.-fSi NP was immobilized on the wells of microplate and associated directly with the optical transducer of microplate reader. Immobilized biohybrid of Sphingomonas sp.-fSi NP was characterized using SEM. A detection range of 0.1-1ppm MP was achieved from the linear range of calibration plot. After integration with fSi NP the storage stability of biohybrid was enhanced ten times from 18 to 180 days. This study proves that after interaction of cells with fSi NP, improved the sensitivity and stability of the biosensor. Spiked samples were also analyzed and correlated using this biohybrid based biosensor.

Synthesis of reticulated hollow spheres structure NiCo2S4 and its application in organophosphate pesticides biosensor.

Electrode materials play a key role in the development of electrochemical sensors, particularly enzyme-based biosensors. Here, a novel NiCo2S4 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 NiCo2S4 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 NiCo2S4 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 1.0×10-12-1.0×10-8gmL-1 with the detection limit of 4.2×10-13gmL-1 for methyl parathion, and 1.0×10-13-1.0×10-10gmL-1 with the detection limit of 3.5×10-14gmL-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.

Improved efficiency of a novel methyl parathion hydrolase using consensus approach.

A methyl parathion hydrolase (MPH) gene, bjmpd, was cloned from a newly isolated MP-degrading bacterial strain, Burkholderia jiangsuensis MP-1T and heterologously expressed in Escherichia coli BL21 (DE3). Although the amino acid sequence of the bjmpd-encoded enzyme, named BjMPH, differed from that of MPH from Pseudomonas sp. WBC-3 (PsMPH) in only three residues, Ser132, Val247 and Ala267, a significantly higher specific activity towards MP was exhibited by BjMPH than PsMPH. Among them, Ala267 was identified as a key site affecting the catalytic efficiency, and it was rather conservative (Ala or Ser) in homologous proteins, suggesting that a simple substitution of the residue in conservative site with another conservative residue based on the consensus sequence approach might possibly enhance the catalytic efficiency of the MP-degrading enzyme. Inspired by such an observation, we identified a new mutant, BjMPHT64N, exhibiting 3.78-fold higher catalytic efficiency (kcat/KM) towards MP than its wild-type, reaching 4.20×106M-1s-1. The mutant BjMPHT64N also displayed enhanced reactivities (kcat/KM) towards other organophosphorus pesticides. Homology-modelling analysis indicates that enhanced polar contacts of the 64th residue in this mutant may contribute to stabilizing the structure of the enzyme and promote the interactions between enzyme and substrate. This study generated an efficient MP-degrading enzyme, and provides useful information for enhancing the catalytic efficiency of MPHs via conservative residue substitution based on the consensus approach.

Metabolic Engineering of Pseudomonas putida KT2440 for Complete Mineralization of Methyl Parathion and γ-Hexachlorocyclohexane.

Agricultural soils are often cocontaminated with multiple pesticides. Unfortunately, microorganisms isolated from natural environments do not possess the ability to simultaneously degrade different classes of pesticides. Currently, we can use the approaches of synthetic biology to create a strain endowed with various catabolic pathways that do not exist in a natural microorganism. Here, we describe the metabolic engineering of a biosafety Pseudomonas putida strain KT2440 for complete mineralization of methyl parathion (MP) and γ-hexachlorocyclohexane (γ-HCH) by functional assembly of the MP and γ-HCH mineralization pathways. The engineered strain was genetically stable, and no growth inhibition was observed. Such a strain not only would reduce the toxicity of MP and γ-HCH but also would prevent the accumulation of potentially toxic intermediates in the environment. Furthermore, expression of Vitreoscilla hemoglobin improved the ability of the engineered strain to sequester O2. The inoculation of the engineered strain to soils treated with MP and γ-HCH resulted in a higher degradation rate than in noninoculated soils. Moreover, introduced GFP may be used to monitor the activity of the engineered strain during bioremediation. The engineered strain may be a promising candidate for in situ bioremediation of soil cocontaminated with MP and γ-HCH.

Genetically engineered Pseudomonas putida X3 strain and its potential ability to bioremediate soil microcosms contaminated with methyl parathion and cadmium.

A multifunctional Pseudomonas putida X3 strain was successfully engineered by introducing methyl parathion (MP)-degrading gene and enhanced green fluorescent protein (EGFP) gene in P. putida X4 (CCTCC: 209319). In liquid cultures, the engineered X3 strain utilized MP as sole carbon source for growth and degraded 100 mg L(-1) of MP within 24 h; however, this strain did not further metabolize p-nitrophenol (PNP), an intermediate metabolite of MP. No discrepancy in minimum inhibitory concentrations (MICs) to cadmium (Cd), copper (Cu), zinc (Zn), and cobalt (Co) was observed between the engineered X3 strain and its host strain. The inoculated X3 strain accelerated MP degradation in different polluted soil microcosms with 100 mg MP kg(-1) dry soil and/or 5 mg Cd kg(-1) dry soil; MP was completely eliminated within 40 h. However, the presence of Cd in the early stage of remediation slightly delayed MP degradation. The application of X3 strain in Cd-contaminated soil strongly affected the distribution of Cd fractions and immobilized Cd by reducing bioavailable Cd concentrations with lower soluble/exchangeable Cd and organic-bound Cd. The inoculated X3 strain also colonized and proliferated in various contaminated microcosms. Our results suggested that the engineered X3 strain is a potential bioremediation agent showing competitive advantage in complex contaminated environments.

Secretory expression of organophosphorus hydrolase OPHC2 in Yarrowia lipolytica Polg.

In the present study, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Yarrowia lipolytica and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses showed a major polypeptide band of 36 kDa. The purified enzyme was optimally active at 65°C and pH 8.5 and also displayed good thermal and pH stability using methyl parathion (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate) as a substrate. Moreover, as Y. lipolytica is a non-pathogenic, generally regarded as safe (GRAS) yeast, the cell culture supernatant can be used directly on vegetables and fruits that are contaminated by organophosphorus pesticides.

Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent.

The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0-500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80-118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody.

ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells.

The multi-xenobiotics resistance (MXR) mechanisms are the first line of defense against toxic substances in aquatic organisms and present great importance in the adaptation related to contaminated environments. Methyl parathion (MP) is a widely used organophosphate pesticide, which has been associated to various toxic effects in organisms. In the present work, we studied the main genes related to efflux transporters in zebrafish liver (ZFL) cells exposed to MP with and without an inhibitor of ABC transporters (verapamil). The results concerning transporters activity showed that the MXR mechanism is activated to detoxify from methyl parathion. The toxic effects of MP on ZFL cells were increased in the presence of the efflux transporter inhibitor, once cell viability was significantly decreased in co-exposure experiments. The combined exposure to MP and the inhibitor caused an increase in gene expression of P-gp1 (Abcb1) and MRP4 (Abcc4), suggesting that these transporters isoforms are associated with MP efflux. In general, the expression of genes related to the antioxidant defense system (ADS) was significantly increased in ZFL cells co-exposed to MP and verapamil. These data provide useful insights for better understanding of MP detoxification mechanism in fish hepatocytes.

Design of a macroalgae amperometric biosensor; application to the rapid monitoring of organophosphate insecticides in an agroecosystem.

The immobilization of enzymes onto transducer support is a mature technology and has been successfully implemented to improve biocatalytic processes for diverse applications. However, there exists still need to design more sophisticated and specialized strategies to enhance the functional properties of the biosensors. In this work, a biosensor platform based on innovative fabrication strategy was designed, and employed for the detection of organophosphate (OP) in natural waters. The biosensor was prepared by incorporating acetylcholinesterase enzyme (AChE) to the graphite paste modified with tetracyanoquinodimethane (TCNQ) mediator, along with the use of a macroalgae (Cladaphropsis membranous) as a functional immobilization support. The novel immobilization design resulted in a synergic effect, and led to enhanced stability and sensitivity of the biosensor. The designed biosensor was used to analyze methyl parathion OP insecticide in water samples collected from a demonstrably contaminated lake of São Luis Island, Maranhão, Northeast of Brazil. Water analysis revealed that the aquatic ecosystem was polluted by sub-ppm concentrations of the OP insecticide, and a good correlation was found between values obtained through biosensor and GC-MS techniques. Our results demonstrated that macroalgae-biosensor could be used as a low-cost and sensitive screening method to detect target analyte.