Structure modeling of novel DNA glycosylase enzyme from oral pathogen Streptococcus sanguinis.

The novel 3-methyladenine DNA glycosylase enzyme from oral pathogen Streptococcus sanguinisin involves in DNA repair mechanisms and participates in base excision repair. Its 3D structure is still unknown which may be a potential drug target, therefore here we proposed its putative 3D structure by homology modeling approach. EsyPred3d software produced more precise modeled structure as compare to Swiss model software. The modeled structure was further verified by PROCHECK analysis and subjected to functional site prediction servers for active site residues prediction. The functional site was further validated by molecular docking approach with ligand EDA (3- [2- Deoxyribofuranosyl] - 3H- 1, 3, 4, 5A, 8-Pentaaza- Asindacene-5- monophosphate) from 1F4R. The EDR docked at the cavity of modeled structure of 3-methyladenine DNA glycosylase enzyme with highest Patchdock score of 3966 and lowest Autodock 4 docking energy of -10.30 Kcal/mol. The YA51, LA105, RA107 residues are surrounding the EDA and matching with ligand binding residues predicted by PROFUNC server.

Both authors contributed equally.

Background:
Gram positive bacteria Streptococcus sanguinis is member of the viridans group of streptococci [1].S. sanguinis serves as a tether for the attachment of other oral microorganisms that colonize the tooth surface, form dental plaque, and contribute to development of caries and periodontal disease [2].Pathogenicity of this organism may not remain limited to oral infections but extended to cause life-threatening endovascular disease infective endocarditis, a serious infection of the valves or lining of the heart [3].Like most oral streptococci, this bacterium produces alphahemolysis on blood agar, a characteristic linked to the ability of viridians streptococci to oxidize hemoglobin in erythrocytes by secretion of H 2 O 2 [4].
Advances in field of biotechnology and bioinformatics had accelerated the progress of medical research in combating such diseases.Recently genome of Streptococcus sanguinis had been sequenced [5].Therefore a lot of information regarding Streptococcus sanguinis cellular machinery and tools of its pathogenicity can be elucidated using wealth of knowledge available in data bases.Here we report an explicit approach to model Streptococcus sanguinis putative DNA repair protein 3-methyladenine DNA glycosylase.A potential drug target.The repair mechanism involves replacement of damaged nitrogenous purine and pyrimidine bases [6].Furthermore putative DNA repair proteins possess high degree of sequence conservation with prokaryotic genomes [7].Therefore due to important role of 3-methyladenine DNA glycosylase enzyme in DNA repair mechanism in Streptococcus sanguinis, but lack of its 3D structure motivated us for proposed investigation.The protein sequence for 3methyladenine DNA glycosylase is available on website http://www.sanguinis.mic.vcu.edu/.In the present work, we will develop the putative 3D structure model of 3-methyladenine DNA glycosylase protein from Streptococcus sanguinis by comparative homology modeling method.In addition, we will subject the modeled structure to functional site prediction servers to find putative active site residues which will validate by molecular docking approach.

Methodology:
The genome sequencing of Streptococcus sanguinis has been completed by Virginia Commonwealth University (http://www.sanguinis.mic.vcu.edu).We selected protein sequence coding for putative 3-methyladenine DNA glycosylase enzyme.The structure prediction and analysis was done in following steps: (1) Comparative homology modeling by Swiss model software [8] and ESyPred3D server [9] for obtaining computational 3D model structure for 3-methyladenine DNA glycosylase protein.(2) The energy minimization of modeled structure by GROMOS96 implemented in Swiss model software.(3) Putative functional sites prediction for modeled structure of 3-methyladenine DNA glycosylase enzyme (4) Validation of functional site by molecular docking studies.

Structure Modeling:
The Swiss model is the automated modeling software which develops the 3D structure model of unknown structure protein based on the sequence homology with the known structured protein.It is important to note that for structure prediction, the sequence homology must be higher than 30%.ESyPred3D server predicts the putative 3D modeled structure of 3methyladenine DNA glycosylase via Modeller ( version 6v2) software where it performs the multiple sequence alignments of the query protein sequence (unknown structure ) with known structured protein sequences by using different alignment tools such as Matchbox, Clustal W, Dialign and PSI-BLAST etc.In next step, the best alignment subjected to model building.On the other hand, Swiss model software performs the homology modeling and develops the putative 3D model of 3-methyladenine DNA glycosylase.Here we used default parameters for developing the modeled structure.The 3D modeled structure of 3-methyladenine DNA glycosylase so obtained was further analyzed by PROCHECK software.

Energy Minimization via GROMOS96
After obtaining the putative 3D modeled structure of 3-methyladenine DNA glycosylase enzyme, the structure was subjected for energy minimization step in order to get more optimized structure.The

Validation of functional site
The functional sites finding was further validated by molecular docking via PATCHDOCK [14] and AUTODOCK4 [15] softwars (see Figure 1 for over all methodology).The ligand was extracted from the template proteins, detected in BLAST with lower e value and template pdb matched by Swiss model.The whole modeled protein was taken as centre (without location of any specific amino acid residue) and generated the grid map.

Discussion and Conclusion:
Here we report

Issue 3 Hypothesis
ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 5(3): 136-140 (2010) © 2010 Biomedical Informatics 137 minimization has been done by GROMOS96 force field implemented in Swiss model software.The GROMOS96 helps in minimization of bond stretch energy of the modeled protein.It incorporates both bonded and non bonded form of energy occupied in the protein molecule.Functional site prediction The minimized structure then subjected to different functional site prediction servers PINTS [10], PROFUNC [11], FIRESTAR [12] and Q-SITE FINDER [13] in order to obtain putative active site residues.These servers predict the putative active site residues which may act as ligand binding site or reaction site.

Figure 1 :
Figure 1: Schematic of methodology: (1) Homology modeling of 3-methyladenine DNA glycosylase protein from Streptococcus sanguinis.(2)Procheck analysis of modeled structure (3) Matching with selected template pdb (4)Extraction of ligand from template pdb (5) Screening of ligand against modeled protein (6)Binding analysis of ligand on modeled protein (7) Residues content at 6A 0 of radius with ligand as center (8)Passed modeled structure through functional site prediction servers (9)Predicted functional sites from servers (10)Matching of putative Active site residues from step 7 and step 9.

Table 1 :
3 D model of novel DNA repair protein 3-methyladenine DNA glycosylase from Streptococcus sanguinis whose 3D structure is still unknown using homology modeling.The knowledge gained about the structure of DNA repair protein 3-methyladenine DNA glycosylase from treptococcus sanguinis may be helpful in discovering drugs against this pathogen.The modeled structure by ESyPred3D (Modeller 6v2) showed high accuracy as compare to structure from Swiss model.The structure was further verified by PROCHECK.The energy minimization via GROMOS96 produced optimized structure for the modeled structure.Comparison of Ramachandran plot statistics for modeled structure of 3-methyladenine DNA glycosylase from ESyPred3D (modeler 6v2) and Swiss model software.

Table 2 :
Functional site prediction for modeled 3-methyladenine DNA glycosylase from Streptococcus sanguinis