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IJETST- Vol.||02||Issue||08||Pages 3083-3089||August||ISSN 2348-9480 2015 International Journal of Emerging Trends in Science and Technology DOI: http://dx.doi.org/10.18535/ijetst/v2i8.12 Sequence to Structure Analysis of DOPA Protein from Mucuna pruriens: A Computational Biology Approach 1 ϯ Authors Laxman Nagar * , Anuj Kumar2 ϯ, Vimala. Y1, MNV Prasad Gajula3,* 1 2 Department of Botany, Ch. Charan Singh University, Meerut 250004, UP, India Bioinformatics Infrastructure Facility (BIF), CCS University, Meerut 250004, India 3 Institute of Biotechnology, PJTSAU, Rajendra Nagar, Hyderabad-500030, India Ϯ Equal Contribution *Corresponding Author MNV Prasad Gajula Institute of Biotechnology, PJTSAU, Rajendra Nagar, Hyderabad-500030, India Email: [email protected] Abstract L-DOPA, (L-3, 4-dihydroxyphenylalanine), an anti-nutritional compound is an important intermediate of secondary metabolism in higher plants and is known as a precursor of alkaloids, betalain, melanine, and others. We analyzed the amino acid sequence of DOPA protein from M. pruriens by using computational methods. The DOPA protein M. pruriens was subjected to sequence, structural and functional annotation. Functional domain prediction through Conserved Domain Database (CDD) suggested that DOPA proteins of M. pruriens contain Aspartate aminotransferase (AAT) superfamily associated with various cellular activities. The analysis of predicted secondary structure by PsiPred and SOPMA has shown that high percentage of helices in the protein structure makes DOPA more flexible for folding processes. Sub-cellular localization predictions suggested it to be a cytoplasmic `protein. Homology modeling method was used to deduce the three-dimensional (3D) structure of selected DOPA proteins from M. pruriens. From template search results it has been identified that all the hypothetical proteins share more than 50% sequence identity with crystal structure of Sus scrofa, indicating proteins are evolutionary conserved. Several quality assessment and validation parameters computed indicated that predicted homology models are in accordance with literature. Current study will help researchers to better understanding of the predicted models and refinement. Key words: L-DOPA, Homology Modeling, Secondary Structure. 1. Introduction L-DOPA, (L-3, 4-dihydroxyphenylalanine), an anti-nutritional compound is an important intermediate of secondary metabolism in higher plants and is known as a precursor of alkaloids, betalain, melanine, and others. It is also a precursor of catecholamines in animals and anti-nutritional compounds confer insect and disease resistance to plants [1], [2]. L-DOPA is a potent neurotransmitter precursor that is believed, in part, to be responsible Laxman Nagar et al for the toxicity of the Mucuna seeds [3]. L-DOPA plays a very important role in plant physiology and for the treatment of Parkinson's disease [4]. Plants synthesize great variety of non-protein amino acids, among which L-DOPA, a compound with strong allelopathic activity, stands out [5]. This allelochemical is found in large quantities (1% and 4–7% in the leaves and seeds, respectively) in velvet bean [Mucuna pruriens (L.) var. utiliz], a legume of the Fabaceae family that has a nutritional quality www.ijetst.in Page 7083 IJETST- Vol.||02||Issue||08||Pages 3083-3089||August||ISSN 2348-9480 comparable to the soybean [6], [7]. Velvet bean is often used for soil cover and as silage due to the large amount of organic matter with high digestibility it produces. It is estimated that velvet bean can release about 100–450 kg ha−1 of L-DOPA into the soil. Furthermore, its ability to control weeds and nematodes greatly reduces the need to apply artificial chemicals to the crops [6], [8], [9]. In addition, its cultivation in tropical areas is aimed at enriching the soil due to its ability to fix nitrogen [10]. In our study we have considered M. pruriens, which is considered to be one of the most popular Indian medicinal plant, traditionaly used in Ayurvedic Indian medicine, for treatment of different diseases including parkinsonism, male infertility, nervous disorders, and also as an aphrodisiac [11]. Due to high protein concentration (23–35%) M. pruriens is considered an important source of various dietary supplements [12], [7]. DOPA from this herbal medicine plant has not been well studied so far at genomic level and the x-ray crystal or NMR structure is also not available in Protein Data Bank (PDB). So we analyzed the amino acid sequence of DOPA protein from M. pruriens by using different bioinformatics algorithms. We predicted the 3D model structure of DOPA from M. pruriens by using homology modeling method. We then studied the stereochemical properties of the modeled structure using Ramachandran plot. We also compared the amino acid sequence with the other DOPA protein sequence reported in different organisms. 2. Methods 2.1 Sequence Retrieval and Functional Domain Analysis DOPA/tyrosine decarboxylase DC1 from M. pruriens was obtained from the Protein sequence database of NCBI (GI: ABK97629). Functional domain analysis was assessed using Conserved Domain Database (CDD) available at NCBI which is a collection of multiple sequence alignment representing protein domains conserved in molecular evolution. It contains the alignment data from protein families’ databases such as Pfam and SMART [13], [14] . Laxman Nagar et al 2015 2.2 Characterization of DOPA/tyrosine decarboxylase DC1 Protein The physicochemical properties such as molecular mass, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (GRAVY) were calculated by ProtParam from EXPASy server (http://www.expasy.org/ ). The TargetP1.1 was used to analyze the subcellular localization of DOPA [15]. In order to know the key residues responsible for catalytic activity of the enzyme, multiple sequence alignment of DOPA with other closely related protein of decarboxylase family having known crystallographic structures was done on ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/ ) 2.3 Secondary structure prediction The secondary structure prediction was done by SOPMA and PsiPred servers [16], [17]. The predicted secondary structural information of the enzyme was considered to improve the target-template alignment and for building conformations for 3D model of the DOPA. 2.4 Model building by Homology Modeling Homology modeling or comparative modeling is the one of the most robust method for protein structure prediction. This method was supplemented for protein structure prediction based on the experimentally determined structure of another homologous protein [18]. The position specific iterated BLAST (PSI-BLAST) against Protein Data Bank (PDB) was carried out to identify their homologous structures based on the maximum identity with high score and lower e-value [19], while 3D structure was predicted using automated Swiss-Model server [20], [21].The modelled 3D structure of DOPA was visualized using UCSF CHIMERA 1.10 [22]. www.ijetst.in Page 7084 IJETST- Vol.||02||Issue||08||Pages 3083-3089||August||ISSN 2348-9480 2.5 Structure validation by SAVES The Ramachandranplot of the modeled structure was calculated by analyzing phi (Φ) and psi (ψ) torsion angles using PROCHECK available on SAVES server (htttp://nihserver.mbi.ucla.edu/SAVES/) [23], [24] , Molprobity, and PSVS server [25], [26]. Further, the ProSA (https://prosa.services.came.sbg.ac.at/prosa.php ) verified the modeled structure (DOPA) structure from X-ray analysis, NMR spectroscopy and other theoretical calculations. The energy criteria of the modeled structures were compared with large set of known protein structures. 3. Results and Discussion 3.1 Sequence Retrieval and Functional Domain Analysis DOPA/tyrosine decarboxylase DC1 from M. pruriens was extracted from the Protein sequence database of NCBI (GI: ABK97629) having 164 amino acids in length. Conserved domains analysis carried by CDD revealed that DOPA protein belongs to Aspartate aminotransferase (AAT) superfamily (Pssm-ID: 276217) with conserved domain length 164, bit score: 224.02 and e-value: 4.89e-71. 3.2 Characterization of DOPA Protein Physiochemical properties of DOPA by ProtParam tools are presented in (Table 1). Results show that DOPA protein has a molecular weight of 17261.1 Daltons and an isoelectric point of 6.78. The computed isoelectric point will be useful for separating the protein on a polyacrylamide gel by isoelectric focusing. The extinction coefficient can be used to calculate the concentration of a protein in solution. Stability of DOPA was studied by analyzing the values for instability index, aliphatic index and Grand average of hydropathicity (GRAVY) index . The value of instability index was 27.38 hence it could be safely predicted as a stable protein. The aliphatic index refers to the relative volume of a protein that is occupied by aliphatic side chains and contributes to the increased thermo stability of protein. Aliphatic Laxman Nagar et al 2015 index of DOPA was 106.52. GRAVY index indicates the solubility of proteins, GRAVY index of DOPA was 0.896. GRAVY value for DOPA describes it to be hydrophilic in nature. Subcellular localization was predicted by TargetP1.1 revealed that DOPA found in the cytoplasm. Table 1. Physicochemical properties of DOPA/ tyrosine decarboxylase DC1predicted by ProtParam program Sr.No 1 2 3 Parameters Molecular Weight Theoretical PI Extinction coefficient* Values 17261.1Dalton 6.78 15720 at Abs 0.1% 0.896 4 Instability Index 27.38 5 Aliphatic index 106.52 6 Grand average of 0.392 hydropathicity(GRAVY) * Extinction Coefficient units M-1cm-1 at 260 nm 3.3 Multiple Sequence Alignment The identification of catalytic residues is a key to understanding the function of proteins. With the information from other functionally similar sequences with known crystallographic structures we can identify the key catalytic residues. ClustalW2 server was used for multiple sequence alignment of DOPA with other decarboxylases from human (PDB Id: 3RBF_A), Human aromatic L- amino acid decarboxylase (PDB Id: 3RCH_A) and Human histidine decarboxylase (PDB Id: 4E1O_A) shown in (Fig.1).The compared sequences varied in length but essentially conserved the key catalytic residues which have been highlighted with an asterisk (*) symbol. www.ijetst.in Page 7085 IJETST- Vol.||02||Issue||08||Pages 3083-3089||August||ISSN 2348-9480 2015 Fig.2. Predicted secondary structure of DOPA protein by using PsiPred Server. Fig.1. Multiple Sequence Alignment (MSA) of DOPA and templates proteins structure using CLUSTAL 2.1. 3.4 Secondary Structure Prediction Secondary structure was predicted by PsiPred and SOPMA servers (Fig.2). The results from SOPMA are shown in (Table 2). Table 2. Secondary structure elements as predicted by SOPMA for DOPA/ tyrosine decarboxylase DC1 Secondary No of amino Percentage structure acid involved Element Alpha helix 76 46.34% Extended 29 17.68% strand Beta turn 10 6.10% Random coil 49 29.88% 3.5 Homology modeling of DOPA Protein We selected a template from Protein data bank (PDB) as a result of PSI BLAST which showed 56% similarity (PDB ID: 1JS3, Chain A, Crystal structure of DOPA decarboxylase in complex with the inhibitor carbiDOPA, X-ray, Minimized Mean Structure, Resolution: 2.25Å) to build 3D structure of M. pruriens DOPA using automate homology modeling server Swiss-Model (Biasini et al. 2014). Generated model showing the closest Cα RMSD (root-mean-square deviation) with respect to their template upon superposition were selected for further structural characterization. Generated model was visualized by UCSF CHIMERA 1.10. (Fig. 3 (A)) High percentage of helices in the structure makes the protein more flexible for folding, which might increase protein interactions. Moreover the predicted secondary structural information of DOPA was considered to improve the target-template alignment and for building 3D model of the DOPA. Fig.3. Structure to validation: (A): Predicted 3D model of DOPA protein; (B): Ramachandran plot for predicted DOPA 3D model; and (C): ProSA plot of DOPA protein Laxman Nagar et al www.ijetst.in Page 7086 IJETST- Vol.||02||Issue||08||Pages 3083-3089||August||ISSN 2348-9480 3.6 Structure Validation Ramachandran dihedral statistics for DOPA revealed a total of 88.7%, 9.2 %, 0.7% and 1.4% residues in most favored, additionally allowed, generously allowed and disallowed regions, respectively. The Ramachandran plot generated by PROCHEK showed the satisfactory geometry of the predicted model (Fig. 3 (B)), (Table. 3). PSVS predicted G- factor score (all dihedral angles) -0.11 revaled that model was 2015 acceptable. Verified3D Z-score was -2.85 suggested that generated model have good quality. ProsaII (-ve) and MolProbity predicted. The Z-score -2.15 and -9.27 also suggested a good model. S. Similar studies were performed by Anuj et al. has shown that the predicted models can be used for further research [27. 28]. ProSA predicted Z-score value was -5.00, evidencing highly reliable structure (Fig.3 (C)). (Table. 3) Table 3. Summary of structure quality factors of DOPA predicted by using SAVES and PSVS 1.5 server Algorithm Mean SD Z-score g score Procheck G-factor e(phi / psi -0.14 N/A -0.24 only) Procheck G-factor e(all dihedral -0.11 N/A -1.65 angles) Verify3D 0.28 0.0000 -2.89 ProsaII (-ve) 0.13 0.0000 -2.15 MolProbity clash score Ramachandran Plot Summary from Procheck Most favoured regions Additionally allowed regions Generously allowed regions Disallowed regions 62.91 0.0000 -9.27 88.7% 9.2% 0.7% 1.4% f Residues selected based on: Dihedral angle order parameter, with S(phi)+S(psi)>=1.8 Selected residue ranges: 1A-163A g With respect to mean and standard deviation for a set of 252 X-ray structures < 500 residues, of resolution <= 1.80 Å, R-factor <= 0.25 and R-free <= 0.28; a positive value indicates a 'better' score 4. Conclusion In this present study, we have used sequence analysis, secondary structure analysis, functional domain prediction and structure prediction to assign to DOPA protein from M. pruriens. L-DOPA is the drug of choice in the treatment of Parkinsonism disease. This study will lead for further research in optimizing the functionality of DOPA which has large implications in treatment of various human diseases. Experimental validation through X-ray crystallography and / or any other spectroscopic techniques can provide more insight into the actual function of this protein. Laxman Nagar et al Acknowledgment Authors acknowledge DST for the Ramanujan fellowship award SRN/S2/RJN-22/2011. References 1. Misawa. M. (1994). Products of interest of industry. In Plant Tissue Culture: An Alternative for Production of Useful Metabolites (pp. 45-69). Bio International, Canada Inc. Toronto. 2. D'Mello, J. P. F., (1995). Leguminous leaf meals in non-ruminant nutrition. 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