current trends in biotechnology and pharmacy vol. 10 (3...

Current Trends in Biotechnology and PharmacyVol. 10 (3) 222-228 July 2016, ISSN 0973-8916 (Print), 2230-7303 (Online)

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AbstractEndoxylanase is involved in the

degradation of xylan to xylose. It majorly breaksdown the hemicelluloses, which are the majorcomponents of the cell wall of the plants. In thepresent study the 3D modeling of endoxylanasefrom Trichoderma pseudokoningii (B0FXM0) wasperformed by comparing modeling approachusing endo 1-4 β-xylanase from Trichodermareesei (PDB ID: 1XYP) as template inmodeler7V.7 program server. The best modelwas selected based on overall stereo-chemicalquality (PROCHECK, proSA, Verified_3D), andalso verified with Protein Structure Validation Suit(PSVS). The active sites of endoxylanase wereidentified in CastP server, which showed that therefined model has highly conserved residues.

Key words: Homology modeling, Modeller,Ramachandran plot, Xylanase.

IntroductionXylan is structural component of cell wall

polysaccharides and it is formed by xylosesubunit which is linked together by β-1,4-glycosidic linkages. The complete hydrolysis ofxylan requires xylanolytic enzymes which areproduced by Trichoderma spp. These are free-living fungi, they are highly interactive in plantroots, soil and foliar environments and theycommercially act as biological control agents(BCAs) against fungal pathogens (1,2).Trichoderma has many industrial applications likeproduction of hydrolytic enzymes such as

xylanases and cellulases (3), and production ofethanol (4). Xylanases are potentially used inwood pulp, fruit juice clarification (5) andsaccharification of agricultural residues (6). Theligno-cellulases (xylanases and cellulases) areclassified into 9 families (A, B, C, D, E, F, G, Hand I) based on the hydrophobic cluster analysisand sequence similarly (7). Based on theclassification of xylanases, they come under For G (8, 9). Xylanase hydrolyses xylan into xylo-oligosaccharides and xylose, similar to that oflysozyme, act by retention of anomericconfiguration, with two glutamate residues beinginvolved in the catalytic mechanism (10). The firstresidue is an acid catalyst that protonating theoxygen of the glycosidic bond, splitting twocellulose or hemi-cellulose subunits and formingan oxocarbonium intermediate. The secondresidue acts as a nucleophilic attack, which bindswith the oxocarbonium intermediate andpromotes the formation of an OH-form a watermolecule, which converts the intermediate into afree xylobiose subunit (10).

The homology modeling of enzymes canoften provide invaluable information. The X-raydiffraction is commonly used strategy to studythe substrates binding sites of enzymes (11). Inabsence of crystallographic structures, a varietyof advanced homology methods have beendeveloped, which can provide reliable models ofproteins that shares 30% or more sequenceidentity with a known structure (12), forunderstanding a structure and function. The

Homology Modeling of Endoxylanase from Trichodermapseudokoningii

Bandikari Ramesh, Seelam Naga Sivudu, Katike Umamahesh andObulam Vijaya Sarathi Reddy*

Department of Biochemistry, Sri Venkateswara University, Tirupati - 517 502, India.*For Correspondence - [email protected]

Endoxylanase homology modeling


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objective of our study was to understand, howthe three dimensional (3D) model ofendoxylanase is generated based on the crystalstructure of the template by using theMODELLER 7v.7, validation of model workflowfollowed in this work given in Fig. 1. These studiesare helpful for the further experimentalinvestigations.

Materials and MethodsSequence retrieval alignment and homologymodeling : The 3D model of xylanase was builtby homology modeling based on high-resolutioncrystal structure of homologous proteins. Thecomplete amino acid sequence of the targetprotein (Endoxylanase) from Trichodermapseudokoningii (B0FXM0) was retrieved fromuniport sequence database in FASTA format(http://www.uniprot.org/). The NCBI Basic LocalAlignment Search Tool (BLAST) (13,14), for thesequence similarities was used for searching thecrystal structure of the closest homologsavailable in the Brookhaven Protein Data Bank(PDB). Based on maximum identity with highscore and lower e-value, xylanase fromTrichoderma reesei (PDB code: 1XYP) with aresolution of 1.5A was used as template. Querycover was 84% over the length of 192 residues.The 1XYP was used as a template to build themodel by pair-wise alignment using Clustal-Wsoftware.

Three dimensional structure generation andvalidation : The academic version ofMODELLER 7v.7 (http://www.salilab.org/modeller) was used for 3D structure generationbased on the information obtained fromsequence alignment (15), Based on homologymodeling method, the assumption that thestructure of unknown protein will be similar tothe known structure of some reference proteinout of the five models generated by MODELLER(16,17).

Validation of 3D structure : The validation ofstructure of model was performed by inspectionof the ψ and φ distributions of the Ramchandranplot obtained from PROCHECK (18) analysis, theenvironment profile was inspected by usingERRAT graph (structure evaluation server) (17)and the compatibility of model with sequencewere measured by VERIFY_3D (19). The stereo-chemical quality of protein structure with best G-score was measured by using protein structurevalidation suite (PSVS) programme server (16).A high verify 3D profile score indicates the highquality of model.

Active site identification : Xylanase (B0FXM0)active site was identified by using CastP server(20,21). This program predicts automaticallylocating and measuring protein cavities, basedon precise computational geometry methods.CastP identifies and measures pockets andpocket mouth opening, as well as cavities. Theprogram specifies the atoms lining pockets,pocket openings and buried cavities. Thisprogram server measured the volume and areaof pockets and cavities, and the area andcircumference of mouth opening.

Results and DiscussionHomology modeling of xylanase

Homology modeling is a clear relation ofhomology between the sequences of targetprotein and at least one known structure is found.This approach gives reasonable results basedon assumption that tertiary structures of twoproteins will be similar if, their sequences arerelated (23). Template protein sequence was

Fig1. Flow chart for complete homology modelingand active site prediction of endoxylanases.

Bandikari Ramesh et al


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collected by search in Basic Local AlignmentSearch Tool (BLAST) as a Brookhaven ProteinData Bank (PDB). The template showed 84%query coverage with 2e-134 e-value, the templateshown maximum sequence alignment againstprotein. The protein and template were pair-wisealigned by Clustal-W (Omega 1.2.1) server.Alignment of protein sequences were shown inFig. 2.

Structurally conserved regions (SCRs)for the model and the template were determinedby superimposition of the two structures and

resemble with each other (24). The final stablestructure of the B0FXM0 protein obtained fromthe help of SPDBV, it is evident that homology totemplate protein, which is shown in Fig. 3. Thesecondary structure of protein domain elementswere α-helices from 182 A to 191 A residues andβ-sheets of residues were 36A-40A, 43A-49A,74A-81A, 198A-209A, 102A-111A, 115A-123A,162A-171A, 143A-155A, 133A-140A, 55A-59A,64A-69A, 212A-219A, 89A-99A, 178A-181A.

Validation of xylanase domain : The overallstereo-chemical quality of model was assessedby Procheck. The Ramachandran plot showed91.2% residues in most favorable region, 7.7%in allowed region (Table-1), and 1.1% in additionaland disallowed region. The results revealed thatmajority amino acids are in ψ and φ distributionconsist with right handed α-helix and reliable tobe good quality model (Fig. 4). The G-factorindicates the quality of covalent and bond angledistance, were -0.33° for dihydral, for covalent -0.16° and overall -0.06°. The overall main chainand side chain parameter, as evaluated byPROCHECK, are all very favorable. TheRamchandran plot characteristic and G-factorconfirm the quality of predicted model, and overallquality factor of 70.681 in ERRAT graph (Fig. 5)indicates acceptable protein environment, asscores of >50 are acceptable for a reasonablemodel.

In order to investigate the interaction energyof each residue with the remainder of the proteinis negative, a second test was done to applyenergy criteria using prosaII energy plot. Theprosa analysis of the model showed maximumresidues to have negative interaction energy andsome residues with positive energy interactionas shown in Fig. 6.

A final test is the packing of each residueas assessed by verify 3D program that representsthe profile obtained with respect to the residues.The compatibility score above zero in the verify-3D graph corresponds to acceptability side chainenvironment (Fig. 7). In verify-3D protein scorefor each residue in 21 residue sliding window

Fig 2. Sequence alignment between protein(B0FXM0) and template (1XYP) in Clustal-W.

multiple sequence alignment. 1XYP used as areference structure for modeling domain.Coordinates from the reference protein(B0FXM0) to the SCRs, structurally variableregions (SVRs), N-termini and C-termini wereassigned to the target sequence based on thesatisfaction of spatial restraints. In theMODELLER software 20 templates weregenerated, among these least energy templatewas selected. All side chains of the model proteinwere set by rotamers. It was observed thatgenerated protein has a maximum similarity withtemplate protein, due to the both proteins wereshowed maximum sequence similarity. In thecase of homology modeling and design 3Dstructure of B0FXM0 protein with templateprotein, it was shown that they structurally



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Fig 3. Final refined structure of xylanase obtainedfrom SPDBV.

Fig 4. Ramachandran plot of xylanase (B0FXM0)homology model by PROCHECK server.

Fig 5. The 3D profile of xylanases modelverified by ERRAT server.

helps to further validating the model (0.74) andscore found to be near to the template.

The quality of model was also assessed bycomparing the predicted structures to theexperimentally showed structures super-imposition and atoms RMSD assessment. Afterthe refinement process, predicted the possibleRMSD (root mean square deviation) deviationfor covalent bonds and covalent angles relativeto the standard dictionary of xylanase (B0FXM0)were 0.019 Å and 2.1º respectively.

Active site identification of xylanase domain: The possible binding sites of B0FXM0 weresearched based on the structural comparison oftemplate with CastP server. The sequence andstructures of B0FXM0 and 1XYP are wellconserved due to their similar biological function.It shown that secondary structures are highlyconserved and the residues, SER 46, TRP 48,ASN74, VAL 76, ASN 101, TYR 103, SER 105,TYR 107, TRP 109, GLU 116, TYR 118, TYR126, PRO 128, SER 129, THR 130, GLY 131,ALA 132, THR 150, ARG 152, GLN 155, PRO156, SER 157, ILE 158, PHE 164, GLN 166, TRP168, TYR 201, ILE 203, ALA 205, GLU 207 andTYR 209 were in Ball like structures in Fig. 8.

ConclusionThe 3D model of xylanase from

Trichoderma pseudokoeningi was constructed inorder to accomplish its molecular modeling andstudy the active sites of enzyme. The model wasvalidating and it was reliable. In future this workexplores docking studies of xylanases in silico

Table 1. % of residues falling in the core region ofRamachandran’s plot



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Fig 6. PROSA energy plot calculated for the B0FXM0 homology model.

Fig 8. Active site of xylanase model (Ball like structures indicates the active site regions).

Fig 7. The 3D profiles verified results of B0FXM0 homology model by verify_3D server.



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and helps to propose the mechanism ofenzymes.

AcknowledgementWe are thankful to Dr. S.C. Basappa,

former Deputy Director and Scientist, CentralFood Technological Research Institute (CFTRI),Mysore, for his encouragement and criticalcomments on the manuscript.

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