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Predictionof TranslycopeneBindingSiteofLycopeneCleavageOxygenaseEnzymeInvolvedinBixinSyntheticPathway: A

Computational Approach

RaghunathSatpathy,RashmiranjanBehera,Rajeshku.Guruand AparajitaPriyadarshinia

DepartmentofBiotechnology,MIRCLAB,MITSEngineeringCollege,Rayagada,Odisha,India-765017.

INTRODUCTION

MATERIALSAND METHODSSequence retrievaland 3D model building

So due to highly commercial and pharmaceutical importance of the compoundthecloning andprotein engineering approach could be used to enhance thebixinproduction in industrial level.The first enzyme i.e. Lycopene cleavage oxygenase that is involved in the bixin production pathway has beenconsidered for thepresent case

The synthetic pathway of bixin production was obtained from the Metacyc pathway database [6].The sequence for the

Annatto ( L.) contains pigment contains bixin and norbixin, valuable natural colorants [1].These pigments are widely used for industrial food and beverages, cosmetics, pharmaceutical products, and as natural dyesfor textiles [2, 3]. Restrictions are imposed on the use of synthetic additives in the food industry and investigation on

biochemical properties of annatto plantsand seeds as it is havingtumor inhibiting capacity [4].The natural dye bixin isextracted from the seed of the annatto plant.Bixin is produced from the sub pathway of isoprenoid biosynthesis. Morespecifically from the translycopene the bixin synthetic pathway diverges by the enzyme Lycopene cleavageoxygenase and further the action of intermediate enzymes like Lycopene beta cyclase and Lycopene epsilon cyclaselead to production of the pigment [5].

. The present study aim to model and to find out the translycopene binding site of the 3Dstructure of Lycopene cleavage oxygenase by computational approach whose structure has not been reported yet.Then to check the reliability of the model and validation purposes various tools and molecular dynamics method isused. The combined docking study and automated prediction server has been used to trace the substrate binding

pocket andthe residues present in the translycopene binding site of theenzyme.

Bixa orellana

ASIANJ.EXP.BIOL.SCI.VOL 2(1)2011:47-52

ORIGINAL ARTICLE

ABSTRACT

KEYWORDS

Bixin is a natural dye and a high commercial important compound, produced from Bixin synthetic pathway in case of Bixaorellanaplant. The particularenzyme Lycopenecleavage Oxygenase catalyzes the first stepof reaction pathways from Trans-lycopene to Bixin synthesis. The 3D structure of the enzyme was predicted by MODELLER9v7 program. Model validationwas done by using the output of PROCHECK, DOPE score. The Ramachandran plot for the model was observed as 87.3

percentages of residues is in favourable regions that indicate the model is reliable. The docking study was performed with the substrate Translycopene by AUTODOCK 4.0.2 tool. The binding residues were predicted by CAST P server as GLU 28, ASP 30, LYS 57, THR 59, SER 68, PHE 75, SER 76, GLN 81, PRO 82, and TYR 83. Further stability was realised by computing energy value and RMSD (Root men square deviation) fluctuation of Carbon alpha back bone of the enzyme and the enzymewithsubstratecomplex,by performing moleculardynamicssimulation usingGROMACS tool.

:Homologymodelling,moleculardynamics,Bixin,modelvalidation,docking,Substratebindingsite

ASIANJ.EXP.BIOL.SCI.VOl2(1)2011 47



Lycopene cleavage oxygenase enzyme was retrieved from Swissprot database from Expasy server [7] and a protein

BLAST search was performed against PDB (Protein Databank) to retrieve the corresponding template for the enzyme

[8]. The model was built by MODELLER9v7 program [9]. The MODELLER program uses an automated approach to

comparative protein structure modelling by satisfaction of spatial restraints. In brief the modelling procedure begins

with an alignmentof thesequence to be modelled (target) with related known 3D structures (templates).Theoutput is a

3D model for thetarget sequence containing allmain chain andside chain non-hydrogenatoms [10]. The program alsoemploys probability density functions (PDFs) derived analytically using statistical mechanics and empirically using a

database of known protein structuresas the spatial restraints.

The MODELLER generated structure was analysed for any missing side chains during the process of modelconstruction by SCWRL4 tool [11] and also further verified by PROCHECK [12].The PROCHECK program

provides the information about the stereo chemical quality of a given protein structure and was used to generateRamachandrans plot. The DOPE (discrete optimized potential energy) score of modeller output per residues of themodel was observed. DOPE score is calculated by Modeller program which is the distance dependent statistical

potential based on probabilistic theory. This is extremely useful in making decisions about reliability [13].

In-silico docking was performed with the validated model and energy minimized translycopene structure [14]. For

that AUTODOCK 4.0.2 tool was used which is designed to predict how small molecules, such as substrates or drugcandidates, bind to a receptor of known 3D structure [15].To cross verify the binding site the model was also observedin CASTp server [16] to figure out the substrate binding pocket regions of the enzyme. CASTp provides identificationand measurements of surface accessible pockets as well as interior inaccessible cavities, for proteins and other moleculesalso theaccessible surface area and volume of each pocketand cavity.

The stability of the model was realised by performing molecular dynamics simulation in by GROMACS 4.0.4software in a high performance computing environment. The computing facility utilised is High performance cluster for Biological Applications which is based on Intel Xeon Dual Quad core as processor, Gluster HPC 1.3 X86-64 bitedition ,total 16 nodes each having 4GB of memory. For the calculation of RMSD (Root mean square deviation) andEnergy value of the model protein GROMACS 4.0.4 (Groningen Machine for Chemical Simulation) was used [17].GROMACS is a versatile package to perform molecular dynamics, i.e. simulate the Newtonian equations of motionfor systems with hundreds to millions of particles [18]. Both the model and complex were subjected to molecular

dynamics simulation in water at 300 K temperature and for 2000 Pico second by using Gromos 43a1 force field of GROMACS package.

The considered suitable template for Lycopene cleavage oxygenase protein is 2BIW chain A [19] obtained fromBLAST search against PDB (Protein Data bank). Then Modeler9v7 program was used to generate the threedimensional structure (Figure 1). Modelling was performed for 10 times and the output of the model having lowestmolpdf value was chosen as the final model. The model was observed that contains 369 amino acid residues with 5helixes, 29 strands and 40 numbers of turns. To verify further the predicted structures, The DOPE score for the modelwas obtained from Modeller output has been shown in Figure 2. From the figure the peak indicates that there is nodefect in the loop regions of the model. The validation of the model was carried out using Ramachandran plotcalculations computed with the PROCHECK program. The Phi and Psi distributions of the Ramachandran plot of non-Glycine, non-Proline residues are summarized in Figure 3. Altogether 100% of the residues were in favoured andallowed regions. The overall G-factor (Goodness factor) used was computed as -0.04 (Table 1). The Homologymodelling and validation results obtained were quite satisfactory.

Docking was performed between the Enzyme model as receptor and Translycopene (Ligand) byAutodock 4.0.2 Tool.After Docking Was finished the result .dlg file was analysed to find the best ligand conformation. The docking energy(binding energy +intermolecular energy) was calculated as -8.73. Also from the complex the binding site wasobserved. Castp server was used to find the binding pocket of ligand and receptor. From the Castp server the binding

Modelvalidation

Docking study and prediction of substratebinding site

Moleculardynamics simulationby usingHigh performanceComputing facility

RESULTS AND DISCUSSIONSHomology modelling of Lycopene cleavage oxygenase and modelvalidation

Docking study and prediction of substratebinding site

Predictionoftranslycopenebindingsite ………………….......................................................................….. A computationalapproachR.Satpathy etal

ASIANJ.EXP.BIOL.SCI.VOl2(1)201148



site was predicted that contains residues like GLU 28, ASP 30, LYS 57, THR 59, SER 68, PHE 75, SER 76, GLN 81,PRO 82, TYR 83, (Figure 4). The docking study of the substrate with the enzyme model show the binding residueswhich are consistent with theresult obtained from automatedligand binding pocketprediction tool CASTp.

To check the stability of the enzyme model and enzyme with substrate after docking was subjected to molecular dynamics simulation for 2000 Pico seconds which is a reliable method [20].The molecular dynamics simulation was

performed also both energy and root mean square deviation plots were derived from the respective trajectory file byGromacs software output. The total energy for the complex was observed within the range from -4.85e+05 to -4.8e+05 KJ/mole and total energy for the enzyme model was from -4.75e+05 to -4.65e+05 KJ/mole (Figure 6). TheRMSD fluctuation plot shows the C-alpha backbone deviation during the simulation process is within the range 0.15-0.34 nanometre (Figure 5). The result for root mean square deviation for the enzyme and complex indicates there isvery little deviation in the back bones during molecular dynamics simulation. Since it is a tolerable fluctuation in the

backbone hence it confirms the model protein is stable. Also total energy calculation indicates the complex shows lessnegative value (more stable) in comparison to energy of model alone. So the combined observation of root meansquare deviation and energy provides strong evidence that the substrate translycopene has been lodged in its proper

binding pocket of the enzyme Lycopene cleavage oxygenase.

Moleculardynamics simulationstudy

Table1Ramachandransplotcalculationson3DmodelofLycopenecleavageoxygenaseproteincomputedwiththePROCHECKprogram

Figure1 ThefinalconsideredmodelofLycopenecleavageoxygenasefromModeller output

% residues in favourable regions 87.3

% residues in additional residue regions 9.7

% residues in generously regions 2.3

% residues in disallowed regions 0.6

% of Non Proline and non Glycine residues 100





Figure2 AnalysisoftheDOPEscoreprofileofthemodelobtainedfromModeller program.

Figure3Ramachandranplotcalculationofthepsi/phiangledistributionofthemodelascomputedbyPROCHECK program.

Figure4Predictedsubstratebindingpocketandresiduesofsubstratetranslycopene(Greencolour)





Figure5Rootmeansquaredeviationplot(Carbon Alphabackbone)obtainedfromGromacstoolduringmoleculardynamicssimulationfor 2000Picosecond.

Figure6Energyplotobtainedduring2000Picosecondmolecular dynamicssimulationofthemodelandcomplexat300KtemperaturebyGromacstool.

CONCLUSIONThe work basically describes a working algorithm to predict the substrate binding pockets and residues of an enzyme.Here in this work, we propose a valid and stable 3D model of Lycopene cleavage oxygenase enzyme whose structureis notpresent in thedatabase.Positiveresults obtained from model validation process confirms ourprediction.Also wehave predicted the key residues and substrate binding pocket of the substrate Translycopene. The docking result isfound to be consistent with the CastP server. The RMSD and energy calculation for the enzyme model and theenzyme- substrate complex by molecular dynamics study shows the validity of the predicted binding site. However this is a computational work further experimental analysis is needed and by using protein engineering approach it is

possible to enhance the production of thecommercially importantcompoundi.e. Bixin.





ACKNOWLEDGEMENT

REFERENCES

We are thankful to Chief executive and Dean of Majhighariani Institute of Technology & Science, Rayagada for providing us MIRC lab for computing facility.

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Corresponding Author: RaghunathSatpathy, DepartmentofBiotechnology,MIRCLAB,MITSEngineeringCollege,

Rayagada,Odisha,India-765017. E-mail:[email protected]



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