The Human Paraoxonase 2: An Optimized Procedure for Refolding and Stabilization Facilitates Enzyme Analyses and a Proteomics Approach.

The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.

Insights in detection and analysis of organophosphates using organophosphorus acid anhydrolases (OPAA) enzyme-based biosensors.

The human population is dependent on agriculture for its food requirements and survival. Several insecticides and pesticides have found their use for improvements in agricultural yields. Organophosphates (OP) are one of the many compounds used as insecticides and pesticides. OPs have also been used to develop G and V-series chemicals which act as highly toxic nerve agents that can severely influence the normal function of the nervous system in all living beings. Thus, OP compounds utilized as insecticides/pesticides and nerve agents are hazardous to the environment, lethal for humans and other non-target animals. To avoid their toxicity, approaches to detect and neutralize them have become essential. A variety of analytical procedures such as electrochemical processes and chromatography methods, namely liquid and gas chromatography, have been employed to detect OPs. Though these techniques are sensitive and highly accurate they suffer from drawbacks, for instance: their bulky nature and expensive instrumentation, the difficulty of operation, long detection times, and they can yield unpredictable results with variable sample complexities. With the advent of several types of biosensors, the assay of OP compounds has become simpler, faster, cost-effective with improved sensitivity, and provides the capability for onsite detection. OP biosensor assays typically utilize several enzymes with the capability to hydrolyze/degrade OP compounds, such as organophosphate hydrolase (OPH) and organophosphate acid hydrolase (OPAA). This review focuses on discussing various aspects of OPAA as biological recognition unit in terms of its: structure, properties, activity enhancement methods, and utilization for developing OPAA-based biosensing technologies for insecticides, pesticides, and nerve agents.

Evaluation of in vitro effect, molecular docking, and molecular dynamics simulations of some dihydropyridine-class calcium channel blockers on human serum paraoxonase 1 (hPON1) enzyme activity.

Paraoxonase 1 (PON1) was purified 148.80-fold in 37.92% yield by hydrophobic interaction chromatography technique. The purity of PON1 was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a single band of 43 kDa. The in vitro effects of nine different calcium channel blockers on PON1 activity were evaluated. All drugs strongly decreased PON1 activity, and IC50 levels were between 13.987 ± 0.59 and 238.104 ± 2.14 µM, Ki values between 8.58 ± 0.36 and 111 ± 1.27 µM. The drugs with the strongest inhibitory effect were nisoldipine with 13.987 ± 0.59 µM and nicardipine with 20.158 ± 0.43 µM. The mechanism of action for the inhibition of the enzyme by nisoldipine and nicardipine was investigated through molecular docking. The stability of enzyme-ligand complexes obtained from the docking was explored through molecular dynamics simulation. The binding affinity of the ligands toward the enzyme was also investigated through MMPBSA (molecular mechanics Poisson-Boltzmann surface area method). The computational analysis demonstrated these compounds could inhibit the enzyme. Nisoldipine had the strongest binding, and its complex was the most stable one. Furthermore, nicardipine was found to have the highest affinity toward the enzyme.

Self-Assembling Enzymatic Nanocomplexes with Polypeptides and Low-Weight Organic Compounds: Preparation, Characterization, and Application of New Antibacterials.

The self-assembling of nanosized materials is a promising field for research and development. Multiple approaches are applied to obtain inorganic, organic and composite nanomaterials with different functionality. In the present work, self-assembling nanocomplexes (NCs) were prepared on the basis of enzymes and polypeptides followed by the investigation of the influence of low-molecular weight biologically active compounds on the properties of the NCs. For that, the initially possible formation of catalytically active self-assembling NCs of four hydrolytic enzymes with nine effectors was screened via molecular modeling. It allowed the selection of two enzymes (hexahistidine-tagged organophosphorus hydrolase and penicillin acylase) and two compounds (emodin and naringenin) having biological activity. Further, such NCs based on surface-modified enzymes were characterized by a batch of physical and biochemical methods. At least three NCs containing emodin and enzyme (His6-OPH and/or penicillin acylase) have been shown to significantly improve the antibacterial activity of colistin and, to a lesser extent, polymyxin B towards both Gram-positive bacteria (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli).

Structural Characteristics of PON1 with Leu55Met and Gln192Arg Variants Influencing Oxidative-Stress-Related Diseases: An Integrated Molecular Modeling and Dynamics Study.

Background and Objectives: PON1 is a multi-functional antioxidant protein that hydrolyzes a variety of endogenous and exogenous substrates in the human system. Growing evidence suggests that the Leu55Met and Gln192Arg substitutions alter PON1 activity and are linked with a variety of oxidative-stress-related diseases. Materials and Methods: We implemented structural modeling and molecular dynamics (MD) simulation along with essential dynamics of PON1 and molecular docking with their endogenous (n = 4) and exogenous (n = 6) substrates to gain insights into conformational changes and binding affinity in order to characterize the specific functional ramifications of PON1 variants. Results: The Leu55Met variation had a higher root mean square deviation (0.249 nm) than the wild type (0.216 nm) and Gln192Arg (0.202 nm), implying increased protein flexibility. Furthermore, the essential dynamics analysis confirms the structural change in PON1 with Leu55Met vs. Gln192Arg and wild type. Additionally, PON1 with Leu55Met causes local conformational alterations at the substrate binding site, leading to changes in binding affinity with their substrates. Conclusions: Our findings highlight the structural consequences of the variants, which would increase understanding of the role of PON1 in the pathogenesis of oxidative-stress-related diseases, as well as the management of endogenous and exogenous chemicals in the treatment of diseases.

Ophthalmic drugs: in vitro paraoxonase 1 inhibition and molecular docking studies.

Glaucoma is a neuropathy disorder and is generally treated by drugs. Allergic conjunctivitis is a common ophthalmologic disease. Paraoxonase 1 (PON1) is an organophosphate hydrolyzer and antiatherogenic enzyme. PON1 is known for preventing atherosclerosis through lipid-modifying features, as well as which has decisive actions of antiapoptosis, anti-inflammatory, antithrombosis, and antiadhesion antioxidant activity properties. Thus, reducing the enzyme levels in hyperthyroidism, chronic renal failure, glaucoma, diabetes mellitus, and cardiovascular diseases is a significant risk. This study was tested some ophthalmic drugs used to treat the diseases, such as glaucoma and allergic conjunctivitis, mentioned above, travoprost, latanoprost, ketotifen, emedastine, and olopatadine, for their inhibition activities against PON1. These drugs displayed the potent inhibition effect with IC50 values ranging between 14.95 ± 0.15 and 299.60 ± 4.07 µM and KI constants ranging from 9.71 ± 2.63 to 261.50 ± 59.98 µM. Besides, the molecular docking analyses of the competitive inhibitors, travoprost, emedastine, and olopatadine, were performed to understand the binding interactions on the enzyme's binding site. According to both in vitro and in silico analysis results, travoprost had the most potent effect on PON1 enzyme activity.

Combined Modification of Fiber Materials by Enzymes and Metal Nanoparticles for Chemical and Biological Protection.

To obtain fiber materials with pronounced chemical-biological protection, metal (Zn or Ta) nanoparticles were jointly applied with polyelectrolyte complexes of enzymes and polypeptides being their stabilizers. Computer modeling revealed the preferences between certain polyelectrolyte partners for N-acyl-homoserine lactone acylase and hexahistidine-tagged organophosphorus hydrolase (His6-OPH) possessing the quorum quenching (QQ) behavior with bacterial cells. The combinations of metal nanoparticles and enzymes appeared to function better as compared to the combinations of the same QQ-enzymes with antibiotics (polymyxins), making it possible to decrease the applied quantities by orders of magnitude while giving the same effect. The elimination of Gram-positive and Gram-negative bacterial cells from doubly modified fiber materials notably increased (up to 2.9-fold), whereas His6-OPH retained its hydrolytic activity in reaction with organophosphorus compounds (up to 74% of initially applied activity). Materials with the certain enzyme and Zn nanoparticles were more efficient against Bacillus subtilis cells (up to 2.1-fold), and Ta nanoparticles acted preferentially against Escherichia coli (up to 1.5-fold). Some materials were proved to be more suitable for combined modification by metal nanoparticles and His6-OPH complexes as antimicrobial protectants.

The Removal of Time-Concentration Data Points from Progress Curves Improves the Determination of Km: The Example of Paraoxonase 1.

Several approaches for determining an enzyme's kinetic parameter Km (Michaelis constant) from progress curves have been developed in recent decades. In the present article, we compare different approaches on a set of experimental measurements of lactonase activity of paraoxonase 1 (PON1): (1) a differential-equation-based Michaelis-Menten (MM) reaction model in the program Dynafit; (2) an integrated MM rate equation, based on an approximation of the Lambert W function, in the program GraphPad Prism; (3) various techniques based on initial rates; and (4) the novel program "iFIT", based on a method that removes data points outside the area of maximum curvature from the progress curve, before analysis with the integrated MM rate equation. We concluded that the integrated MM rate equation alone does not determine kinetic parameters precisely enough; however, when coupled with a method that removes data points (e.g., iFIT), it is highly precise. The results of iFIT are comparable to the results of Dynafit and outperform those of the approach with initial rates or with fitting the entire progress curve in GraphPad Prism; however, iFIT is simpler to use and does not require inputting a reaction mechanism. Removing unnecessary points from progress curves and focusing on the area around the maximum curvature is highly advised for all researchers determining Km values from progress curves.

Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site.

Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.

Endothelial Lipase Modulates Paraoxonase 1 Content and Arylesterase Activity of HDL.

Endothelial lipase (EL) is a strong modulator of the high-density lipoprotein (HDL) structure, composition, and function. Here, we examined the impact of EL on HDL paraoxonase 1 (PON1) content and arylesterase (AE) activity in vitro and in vivo. The incubation of HDL with EL-overexpressing HepG2 cells decreased HDL size, PON1 content, and AE activity. The EL modification of HDL did not diminish the capacity of HDL to associate with PON1 when EL-modified HDL was incubated with PON1-overexpressing cells. The overexpression of EL in mice significantly decreased HDL serum levels but unexpectedly increased HDL PON1 content and HDL AE activity. Enzymatically inactive EL had no effect on the PON1 content of HDL in mice. In healthy subjects, EL serum levels were not significantly correlated with HDL levels. However, HDL PON1 content was positively associated with EL serum levels. The EL-induced changes in the HDL-lipid composition were not linked to the HDL PON1 content. We conclude that primarily, the interaction of enzymatically active EL with HDL, rather than EL-induced alterations in HDL size and composition, causes PON1 displacement from HDL in vitro. In vivo, the EL-mediated reduction of HDL serum levels and the consequently increased PON1-to-HDL ratio in serum increase HDL PON1 content and AE activity in mice. In humans, additional mechanisms appear to underlie the association of EL serum levels and HDL PON1 content.

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions.

Organophosphorus compounds (OPCs) are able to interact with various biological targets in living organisms, including enzymes. The binding of OPCs to enzymes does not always lead to negative consequences for the body itself, since there are a lot of natural biocatalysts that can catalyze the chemical transformations of the OPCs via hydrolysis or oxidation/reduction and thereby provide their detoxification. Some of these enzymes, their structural differences and identity, mechanisms, and specificity of catalytic action are discussed in this work, including results of computational modeling. Phylogenetic analysis of these diverse enzymes was specially realized for this review to emphasize a great area for future development(s) and applications.

The role of paraoxonase in cancer.

The paraoxonase (PON) gene family includes three proteins, PON1, PON2 and PON3. PON1 and PON3 are both associated with high-density lipoprotein (HDL) particles and exert anti-oxidant and anti-inflammatory properties. PON2 and PON3 are intracellular enzymes which modulate mitochondrial superoxide anion production and endoplasmic reticulum (ER) stress-induced apoptosis. The pleiotropic roles exerted by PONs have been mainly investigated in cardiovascular and neurodegenerative diseases. In recent years, overexpression of PON2 and PON3 has been observed in cancer cells and it has been proposed that both enzymes could be involved in tumor survival and stress resistance. Moreover, a lower activity of serum PON1 has been reported in cancer patients. This review summarizes literature data on the role of PONs in human cancers and their potential role as a target for antitumor drugs.

Naphthoquinones, benzoquinones, and anthraquinones: Molecular docking, ADME and inhibition studies on human serum paraoxonase-1 associated with cardiovascular diseases.

Paraoxonase-1 (PON1) has essential roles such as protecting low-density lipoprotein against detoxification and oxidation of highly toxic compounds. Quinones are a class of compounds and a type of plant-derived secondary metabolites. Here, PON1 was purified using very simple methods and evaluation of the interactions between the enzyme and some quinones. It was found that these quinones displayed effective inhibitor properties for PON1 with the IC50 values in the range of 3.27-82.90 µM and the K i values in the range of 2.50 ± 0.65 to 30.90 ± 7.20 µM. These quinones displayed distinct inhibition mechanisms. It was determined that except for 5-hydroxy-2-methyl-1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone all quinones exhibit competitive inhibition effects. Also, molecular docking and in silico ADME studies were performed. Usage of drugs including quinone derivatives in structure with biological activity would be hazardous in some cases.

Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity.

Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 105 M-1 s-1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.

Targeted Heating of Enzyme Systems Based on Photothermal Materials.

This study demonstrates that the enzymatic reaction rate can be increased significantly by targeted heating of the microenvironment around the enzyme, while maintaining the reaction system at environmental temperature. Enzyme molecules are covalently attached to the surface of Fe3 O4 @reduced graphite oxide (rGO). Under visible-light irradiation, the reaction rate catalyzed by the enzyme-Fe3 O4 @rGO system is clearly enhanced relative to that of the free enzyme and a mixture of free enzyme and Fe3 O4 @rGO. This local heating mechanism contributes to promotion of the enzymatic reactions of the targeted heating of the enzyme (THE) system, which has been validated by using different enzymes, including lipase, glucose oxidase, and organophosphorus hydrolase. These results indicate that targeted heating of the catalytic centers has the same effect on speeding up reactions as that of traditional heating methods, which treat the whole reaction system. As an example, it is shown that the THE system promotes the sensitivity of an enzyme screen-printed electrode by 14 times at room temperature, which implies that the THE system can be advantageous in improving enzyme efficiency, especially if heating the entire system is impossible or could lead to degradation of substrates or damage of components, such as in vitro bioanalysis of frangible molecules or in vivo diagnosis.

Identification a novel clinical biomarker in early diagnosis of human non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a malignant tumor with high morbidity and mortality. The clinical biomarkers currently used for the early diagnosis of lung cancer have poor sensitivity and specificity. Therefore, it is urgent to identify sensitive biomarkers for the early detection of NSCLC to improve the patient survival of patients. In our previously study, we identified glycoprotein alpha-1-antichymotrypsin (AACT) as an early biomarker of NSCLC. In this study, serum glycopeptides were enriched using the high-GlcNAc-specific binding lectin, AANL/AAL2, for further quantitative proteomics analysis using LC-MS/MS. A total of 55 differentially expressed proteins were identified by using demethylation labelling proteomics. Serum paraoxonase/arylesterase 1 (PON1) was selected for validation by western blotting and lectin-ELISA in samples from 120 enrolled patients. Our data showed that AANL-enriched PON1 has better diagnostic performance than total PON1 in early NSCLC, since it differed between early Stage I tumor samples and tumor-free samples (healthy and benign). Combining AANL-enriched PON1 with carcinoembryonic antigen (CEA) significantly improved the diagnostic specificity of CEA. Moreover, combined AANL-enriched PON1 and AANL-enriched AACT was significantly different between early NSCLC samples and tumor-free samples with an AUC of 0.940, 94.4% sensitivity, and 90.2% specificity. Our findings suggest that combined AANL-enriched PON1 and AANL-enriched AACT is a potential clinical biomarker for the early diagnosis of NSCLC.

Association of human serum paraoxonase-1 with some respiratory drugs.

In this study, we investigated the effects of certain respiratory drugs, which are mainly used on human serum paraoxonase-1 (hPON1; EC 3.1.8.1). hPON1 was purified from human serum, with 354.91 fold and 45% yield by using two simple step procedures including, first, ammonium sulfate precipitation, then, Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis showed a single protein band belonging to hPON1 with 43 kDa. All the pharmaceutical compounds inhibited the PON1 enzyme highly at the micromolar level. The obtained IC50 values for nine different pharmaceutics ranged from 0.219 µM (salbutamol sulfate) to 67.205 µM (montelukast sodium). So, all drugs could be considered as potent hPON1 inhibitors. Ki values and inhibition types were determined by Lineweaver-Burk graphs. While varenicline tartrate and moxifloxacin hydrochloride inhibited the enzyme in a noncompetitive manner, others inhibited it in a mixed manner.

Organophosphate hydrolase conjugated UiO-66-NH2 MOF based highly sensitive optical detection of methyl parathion.

The hexahistidine-tagged organophosphorus hydrolase (OPH6His) has been immobilized on a Zr-MOF, namely UiO-66-NH2. The resulting enzyme-MOF composite was used as a carrier to facilitate the hydrolysis of an organophosphate pesticide, i.e., methyl parathion in to p-nitrophenol (PNP). The formation of PNP took place in direct proportion to the added pesticide concentration. Coumarin1 (7-diethylamino-4-methylcoumarin) was then introduced in the reaction mixture as a reporter fluorescent molecule. As PNP acted to quench the fluorescence of coumarin1, it became possible to detect methyl parathion over a wide concentration range of 10-106 ng/mL with an achievable limit of quantification as 10 ng/mL. The immobilization of OPH6His on the surface of UiO-66-NH2 was found to endow an improvement in the enzymatic activity by about 37%. The OPH6His/UiO-66-NH2 conjugate was reusable for at least up to eight times and also found stable toward long-term storage (minimum 60 days). The potential practical utility of the above proposed sensing method has been demonstrated by employing it for an accurate analysis of pesticide-spiked food samples.

Evaluation of the substitutions in 212, 342 and 215 amino acid positions in binding site of organophosphorus acid anhydrolase using the molecular docking and laboratory analysis.

OBJECTIVE: Organophosphorus Acid Anhydrolase (OPAA) is used as one of the most important enzymes in the decontamination process of organophosphate compounds. In this study, we aimed to evaluate the effects of amino acid substitution in OPAA's substrate-binding site on its catalytic activity using the rational engineering strategy. METHODS: The native and three mutant forms of OPAA enzyme including 4ZWP, 4ZWU and Mut6 were studied using the docking technique toward parathion, paraoxon and R-VX compounds. Furthermore, enzyme assay was performed on the native OPAA and Mut6 toward parathion. RESULTS: Docking results showed a decreased catalytic activity of the mutant forms toward parathion and paraoxon. Furthermore, enzyme assay showed in accordance with docking results a decreased activity of Mut6 compared to the native form. The results of docking prediction for R-VX showed an increased catalytic activity of 4ZWP and 4ZWU. 4ZWU had the highest activity, while the activity of Mut6 was lower than the native form. CONCLUSION: Amino acid positions of 212 and 342 seem to be important sites in the small pocket of OPAA affecting the enzyme catalytic activity. Therefore, substitution of these sites with appropriate amino acids depending on the substrate structure, can affect the enzyme catalytic efficiency (Tab. 2, Fig. 3, Ref. 30).

Novel approach to quorum quenching: rational design of antibacterials in combination with hexahistidine-tagged organophosphorus hydrolase.

N-acyl homoserine lactones (AHLs) are quorum sensing (QS) signal molecules used by most Gram-negative pathogenic bacteria. In this article the lactonase activity of the preparations based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) towards AHLs was studied. Initially, three of the most interesting ß-lactam antibiotics were selected from seven that were trialed during molecular docking to His6-OPH. Combinations of antibiotics (meropenem, imipenem, ceftriaxone) and His6-OPH taken in the native form or in the form of non-covalent enzyme-polyelectrolyte complexes (EPCs) with poly(glutamic acid) or poly(aspartic acid) were obtained and investigated. The lactonase activity of the preparations was investigated under different physical-chemical conditions in the hydrolysis of AHLs [N-butyryl-D,L-homoserine lactone, N-(3-oxooctanoyl)-D,L-homoserine lactone, N-(3-oxododecanoyl)-L-homoserine lactone]. An increased efficiency of catalytic action and stability of the lactonase activity of His6-OPH was shown for its complexes with antibiotics and was confirmed in trials with bacterial strains. The broadening of the catalytic action of the enzyme against AHLs was revealed in the presence of the meropenem. Results of molecular docking of AHLs to the surface of the His6-OPH dimer in the presence of antibiotics allowed proposing the mechanism of such interference based on a steric repulsion of the carbon chain of hydrolyzed AHLs by the antibiotics bounded to the enzyme surface.