The on the interaction and degradation of chlorpyrifos by

The pesticides has facilitated the development andalso expansion of agriculture in world wide. Organophosphates belong to a classof highly toxic neurotoxins that are commonly used as pesticides and chemicalwarfare agents (Surekha Rani et al. 2008).The continuous use of organophosphates in intensive quantities throughout theworld and their potential neurotoxicity to humans has led to the development ofvarious efficient and safe strategies of bioremediation to deal with their widedispersal in the ecosystem (Cho et al.

2002). Enzymatic degradation byorganophosphorus hydrolase (OPH) has received considerable attention. Thisattention provides the possibility of both eco friendly and in situdetoxification (Catherine et al. 2002). The focus of this work is organophosphorushydrolase (OPH, E.C. 8.1.

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3.1), which catalyzes the hydrolysis of manyorganophosphorus compounds and greatly reduces the toxicity of organophosphatepesticide and even it can completely mineralize them.Identical opd genes coding for OPH were foundin two soil microorganisms, Pseudomonas diminuta MG and Flavobacteriumsp. (Sethunathan et al. 1998). AlthoughOPH hydrolyzes a wide range of organophosphates, the effectiveness ofhydrolysis varies dramatically. Widely used organophosphorus insecticides likemethyl parathion, chlorpyrifos, and diazinon are hydrolyzed slowly than 30 to1,000 times is the preferred substrate, paraoxon (Cho et al.

2002). Thisreduction in catalytic rate is due to the unfavorable interaction of thesesubstrates with the active sites involved in catalysis and/or structuralfunctions (Zheng et al. 2013). A number of enzymes are capable of hydrolysing anumber of organophosphate triesters into less or non-toxic compounds. Theseenzymes are possible bioremediators because of their ability to decontaminateOP-containing waters and soils (Zheng et al. 2013). The most thoroughlycharacterized phosphotriesterases have been isolated from Flavobacterium sp.

ATCC 27551, Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA)(Fernanda et al. 2010). These enzymes belongto the binuclear metallohydrolase family and share high sequence andstructural homology. Phosphotriesterases are highly promiscuous enzymes,hydrolysing a large range of substrates. The phosphotriester hydrolysis by OPHhas been studied extensively (Castro et al. 2016).In a proposed reaction scheme, based on largely crystal structures with boundinhibitors, the phosphoryl oxygen of the substrate binds to the ?-metalion (Janet et al.

2005; Laothanachareo et al. 2008).In the present research focuses on the interactionand degradation of chlorpyrifos by OPH enzyme, as this is responsible fordetoxification. The molecular docking study was conducted under FlexX dockingsoftware package. Materials and methodsIsolation and identification oforganophosphate hydrolase (OPH) producersApotential organophosphate hydrolase(OPH) producing Pseudomonas stutzeriMCAS01 (Kavitha et al. 2016) has been usedin this study. Sequence and template search forhomology modelingPseudomonas stutzeriorganophosphate hydrolase (OPH), 3D structures are not available in ProteinData Bank (PDB) database, the homologous sequences for building the 3Dstructure was searched against PDB using NCBI BLAST (Basic Local AlignmentSearch Tool) (Altschul et al.1990).

Thehomologous sequences are ability template structure for homology modeling. Theatomic coordinate report of the template structure was obtained from the PDB(Berman et al. 2000).Comparative modeling and modelconfirmationTheatomic coordinate file of the template along with the target and template finalsequence alignment file was used to build the model using the automatedhomology modeling tool MODELER 9v9 (Eswar et al.

2006).A bundle of models from the random generation of the starting structure wascalculated and among the generated models, the best model with the least RootMean Square Deviation (RMSD) value was selected by superimposing the model withits template (Maiti et al. 2004).

This modelwas used for further analysis after subjecting it for energy minimization usingGROMOS of Swiss PDB viewer (Walter et al.1999).The quality of the generated model was assessed by checking the stereo chemicalparameters using PROCHECK (Laskowski et al. 1993),Verfiy3D (Bowie et al. 1991; Luthy et al.1992) and ERRAT at SAVES server http://nihserver.mbi.

ucla.edu/SAVES(Colovos et al. 1993).Computational details                All computations were carriedout on an Intel  Core  i3-3240 @ 3.40GHz capacity  processor with  a memory of 8GB RAM runningon windows 7 operating system. Finally docking studies were done using theFlexXdocking software package (https://www.

biosolveit.de/). For the improvement andbinding energy calculations, the default settings of FlexX LeadIT were used.Target proteins and ligands                Protein structure weredownloaded from PDB (PDB id: 3F4D) (Fig. 2)(Hawwa et al. 2009) and the ligand structurewere obtained from pubchem (Pubchem CID: 2730) (Fig.

1) and the functionalinformation of these proteins were retrieved from the Uniprot. Further,hydrogen atoms, bond orders and formal charges were added using the proteinpreparation wizard of the FlexX LeadIT tools as described.Protein and ligand preparation                PDB files of proteins andligands were prepared using FlexX LeadIT protein and ligand preparation wizardand then binding pockets were set using the individual wizard. The interactionsof the ligand with the protein residues in the binding site were visualized.

 StructuralanalysisProteins and ligand interactions were calculated. Acut-off of 1.5 to 3Å distance between the donor and acceptor were used for thecalculation of hydrogen bonds.

All the positive docked sites were generated. Results  Sequence analysisTheorganophosphate hydrolase enzyme producing bacterial strain was identified as Pseudomonas stutzeri through 16s rRNAgene sequence analysis (Kavitha et al. 2016).Sequenced amplicon has been submitted to NCBI database and accession number wasobtained KT757902. The organophousphorushydrolase gene (opd) was sequenced and submitted to the NCBI and the accessionnumber was MG739657. Based on the above information the organophosphatehydrolase sequences were retrieved from PDB for homology modeling.

The BLASTPsearch for target sequences of organophosphate hydrolase from P. stutzeri against the PDB databaseresulted that crystal structure of organophosphate hydrolase was got. Homology modelingThe 3D structure of organophosphate hydrolase from Pseudomonas stutzeri was developed bythe X-ray structure. Modeler 9v9 was used to develop the 3D structure byproviding the alignment file, template file, and target file. The alignmentfile was adjusted by taking into the account of overlap between the secondarystructure elements of the template and the predicted secondary structureprofile of the sequence.

Further, considering the parameter provided for anumber of the model to be calculated as five, modeler provided five initial modelsof cellulose by using random generation and by applying spatial resistance.These generated models were superimposed with a template structure to revealthe degree of modeled structure with the template by calculating the Root MeanSquare Deviation (RMSD). The modeled and energy minimized structure oforganophosphate hydrolase from P.

stutzeri was shown in cartoon representation with group color using rasmolvisualization tool (Fig. 2). Bindingorientation and interactionTheorientation of ligand is important for acceptor binding activity. Clearly,binding orientation of chlorpyrifos model compounds inside the organophosphatehydrolase highly variedas can be seen from Fig.

4, which suggested the performance of organophosphatehydrolase in catalysisfor the degradation of chlorpyrifos model compounds was different. Theinteraction energies were analyzed in detail (Table 1).When making comparisons between these complexes, the most noticeable differencewas the interaction energy. Their interaction energy changed 1 in a wide range.This means that H-bonds were an alternative way to determine the interaction oforganophosphatehydrolase withchlorpyrifos. Hydrophobic interaction seemed to be a more important factor forthe binding of organophosphate hydrolase to chlorpyrifos model compounds thanH-bonds, because all chlorpyrifos model compounds formed hydrophobicinteractions with organophosphate hydrolase (Fig.

3).We observed local differences in the types of amino acid residues participatedin hydrophobic interactions. These results showed that hydrophobic interactionswere necessary for the binding of organophosphatehydrolase tochlorpyrifos model compounds, and thus were potentially important tochlorpyrifos degradation. DiscussionBiodegradationtechnology is becoming more and more attractive for environmental remediationdue to its environmentally friendly nature. An organophosphate hydrolase-basedapplication for chlorpyrifos pesticide degradation is a good example (Singh, 2009). To increase the chlorpyrifos-degradingefficiency of organophosphate hydrolase, previous studies investigated theimpact of substrate structure on organophosphate hydrolase-mediated oxidationrate, the stability of bacterial organophosphate hydrolase, using chlorpyrifosas a model compounds (Fernanda et al.

2010). The chlorpyrifos-degrading efficiencyof organophosphate hydrolase was largely related to the properties of enzymeand substrates, such as their binding property. The stability and catalyticactivities of organophosphate hydrolase was potentially influenced by thebinding modes between it and its substrates. However, the detailed interactionmechanism between organophosphate hydrolase and chlorpyrifos is still unclear,limiting organophosphate hydrolase application in chlorpyrifos degradation tosome extent.

Thus, the illustration of interaction between organophosphatehydrolase and chlorpyrifos model compounds is important. Molecular simulationssuch as molecular docking have proved to be a robust technology for the analysesof intermolecular interactions (Ramalha et al. 2016).

This article performed an investigation of the molecular basis oforganophosphate hydrolase for chlorpyrifos degradation, using moleculardocking. We showed that the present protocol was capable of giving a molecularinsight into the interaction of organophosphate hydrolase with chlorpyrifosmodel compounds, and in this way we found several rules that may be importantto chlorpyrifos degradation, this also states by Jin et al. (2015) Molecular DynamicsSimulations of Acylpeptide Hydrolase Bound to Chlorpyrifosmethyl Oxon andDichlorvos. It was showedthat chlorpyrifos model compounds bound to organophosphate hydrolase by a widerange of interaction energies.

Therefore, we proposed that H-bonds werealternative, but hydrophobic contacts were necessary to the interaction oforganophosphate hydrolase with chlorpyrifos model compounds or chlorpyrifosthis also confirmed by Castro et al. (2016).Mean backbone RMSD values for different complexes varied (Lima et al. 2016). It not only meant stable behavior of thesecomplexes, but also indicated that the stability was different between variouscomplexes. ConclusionAnextended binding analysis was done after docking the Chlorpyrifos against thetargeted protein.

The models created by docking compounds against targetproteins were analyzed and the interactions, hydrogen bonds and distance. Chlorpyrifosefficiently binds to the target protein organophosphate hydrolase with theformation of three hydrogen bonds with residues Asp53, Lys54 and yielded abinding affinity of -5.9124 kcal/mol. Therefore, the present study provides dynamic and structural information onthe interaction mechanism between organophosphate hydrolase and chlorpyrifos,being useful to develop new organophosphate hydrolase with highchlorpyrifos-degrading ability for the prevention of pollution in the soil and  provide an ecofriendly environment.