International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 3(4), 11-17, April (2014) Int. Res. J. Biological Sci. International Science Congress Association 11 In silico analyses of Rubisco Enzymes from different classes of AlgaeBhavisha P. Sheth* and Vrinda S. Thaker The Virtual Institute of Bioinformatics, Department of Biosciences, Saurashtra University, Rajkot 360005, Gujarat, INDIA Available online at: www.isca.in, www.isca.me Received 21st September 2013, revised 24th October 2013, accepted 3rd December 2013Abstract Rubisco (Ribulose 1, 5 Bisphosphate Carboxylase Oxygenase) is the most predominant enzyme of one of the few carbon assimilatory processes in nature i.e. Photosynthesis. The rbcL and rbcS genes code for the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) respectively. In this study the rbcL protein sequences selected from various classes of algae were phylogenetically analyzed. Expasy’s Prot-param server and Cys_rec tool were used for physico-chemical and functional characterization of these proteins. For comparative structural analysis, experimental structures (X-ray and NMR) of rubisco proteins of representative species of Rhodophyta (Galderia sp. PDBID 1IWA) and Chlorophyta (Chlamydomonas sp. 1GK8) were used. Also, as no experimental structure of rubisco from any member of phaeophyta group is available, homology modeling approach was employed in order to derive structure of the same from Lessonia vadosa, a representative species of phaeophyta group. The validity of the modeled protein was further checked by RAMPAGE, Procheck, WHATIF, Errat, and Verify-3d servers. Studies of secondary structure of these proteins were carried out by the SSCP server. The in silico analysis, confirmed the close correlation between the rhodophyte and the phaeophyte rubico proteins at the functional level due to similarity in adaptability of the enzyme. Keywords:rbcL, rubisco, bioinformatics, phylogenetics, protein. Introduction There is a simultaneous increase in the world hunger, with the increase in the global population. Photosynthesis, the process of conversion of light energy to consumable chemical energy, is by far the most predominant of the few carbon assimilatory processes, in contrast to most of the carbon dissimilation processes of nature. Rubisco, responsible for all photosynthetic carbon fixation is the most abundant enzyme on the earth. The properties of rubisco like effectiveness and specificity determine the photosynthetic efficiency and ultimately the productivity of photosynthetic organisms however, is often thought of as a highly conserved and sluggish enzyme. The rbcL gene encodes the large subunit of Rubisco enzyme. As Rubisco catalyzes the rate-limiting step of photosynthesis, the structural basis for catalysis can identify potential targets for useful engineering. However, rubisco enzymes from different species have different catalytic constants. If the structural basis for such differences were known, a rationale could be developed for genetically engineering an improved enzyme. So, the further beneficial genetic engineering of Rubisco may result in substantial increases in crop-plant productivity. The bibliometric and bioinformatic analysis might offer great help in designing better alternatives of the enzyme in silico. Kinetic comparisons of the enzyme with other more potent natural Rubiscos of interest in the evolutionary study will help to authenticate the obtained results. The evolutionary analysis with the integration of bioinformatics tools and experimental validation will bring out the best results in the Rubisco research. The structural analysis of the photosynthetically most efficient, rubisco protein is reported from the red alga. However, no report on brown algae is listed. The present study highlights the correlation between the structural and phylogenetic aspects of the rubisco enzymes, between these major groups of algae i.e. Rhodophyta, Chlorophyta and Phaeophyta and comparison of the same with that of other groups of algae is carried out and the results are discussed. Material and Methods Sequence analyses: Protein sequence phylogenetic analysis: The amino acid sequences of secondary metabolite proteins of various algal rbcL proteinsfrom three representative families viz. Rhodophyceae (Rh) (Rh1-7), Chlorophyceae (Ch) (Ch1-8) and Phaeophyceae (Ph) (Ph1-10) were retrieved from SWISSPROT, a public resource of curated protein sequences (table 1). The multiple sequence alignment of the sequences used in the study was performed in ClustalX. Further, the evolutionary history was inferred using the Neighbor-Joining method and the bootstrap consensus tree was inferred from 2700 replicates using the MEGA5 software (figure 1). Physiochemical characterization: For physiochemical characterization, amino acid composition, theoretical instability index, aliphatic index and GRAVY (grand average hydropathy) were computed using the Expasy’s ProtParam server for set of proteins. The results are shown in table 2 and table 3. Structural analyses: The protein 3D structures of the photosynthetically most efficient alga Galderia partita (PDBID: International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(4), 11-17, April (2014) Int. Res. J. Biological Sci. International Science Congress Association 12 1IWA) and that of a green alga Clamydomonas reinhardtii (PDBID: 1GK8) are retrieved from the RCSB PDB. As experimental structures of any organism from the Phaeophyte group are not available, homology modeling approach was used in order to derive the structure of Lessonia vadosa, a representative of the Phaeophyte group of algae. Homology modelling of Lessonia vadosa Rubisco: The protein sequence of Lessonia vadosa, retrieved from the SwissProt Database was used for BlastP analysis from the NCBI BLAST Suite. Based on high score, lower e-value andmaximum sequence identity, the best template (1IWA) was selected which was then used as reference structure to build a 3D model. The homology model can be seen in figure 2. Model building and evaluation: The three dimensional structures of proteins were modeled using EsyPred Server. Quality of generated models was evaluated with PROCHECK and RAMPAGE server by Ramachandran plot analysis. Validation of generated models was further performed by VERIFY 3D10, WHATIF11 and ERRAT12 programs. The results obtained are shown in figure 3A-D. Submission of the modeled protein in protein model database (PMDB): The model generated for Lessonia vadosa Rubisco was submitted in Protein Model Database, PMDB13. The submitted model can be accessed via its PMID. Ph5 Ph6 Ph3 Ph2 Ph4 Ph8 Ph9 Ph10 Ph7 Ph1 Rh3 Rh4 Rh1 Rh6 Rh2 Rh7 Rh5 Ch4 Ch1 Ch3 Ch2 Ch8 Ch7 Ch5 Ch6 944934232924100989996949995564947100432468 0.05Figure-1 Phylogenetic analysis of rbcL protein sequences from different classes of algae using MEGA5 (Ph1-5: Phaeophyta; Rh1-5: Rhodophyta; Ch1-5: Chlorophyta) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(4), 11-17, April (2014) Int. Res. J. Biological Sci. International Science Congress Association 13 Table-1 List of selected rbcL protein sequences Sr.No Family Code Accession No. Name of organism 1.Rhodophyta Rh1 Q09119 Porphyridium aerugineum 2.Rh2 Q760S7 Porphyra dentata 3.Rh3 P24624 Antithamnion sp. 4.Rh4 P48691 Chondrus crispus 5.Rh5 P23755 Galdieria sulphuraria 6.Rh6 Q760R5 Porphyra haitanensis 7.Rh7 Q760T5 Porphyra yezoensis 8.Chlorophyta Ch1 Q20EX7 Oltmannsiellopsis viridis 9.Ch2 Q0P3J3 Ostreococcus tauri 10.Ch3 P12466 Chlorella vulgaris 11.Ch4 P26958 Bryopsis maxima 12.Ch5 P08211 Chlamydomonas moewusii 13.Ch6 Q2TGZ2 Dunaliella tertiolecta 14.Ch7 B2X1Y2 Oedogonium cardiacum 15.Ch8 Q9T4F2 Nephroselmis olivacea 16.Phaeophyceae Ph1 A1BNI3 Pseudolessonia laminarioides 17.Ph2 A1BNI4 Lessonia nigrescens 18.Ph3 A1BNI5 Lessonia tholiformis 19.Ph4 A1BNI7 Lessonia trabeculata 20.Ph5 A1BNI9 Lessonia vadosa 21.Ph6 A1BNJ1 Lessonia sp. CNUK PL216 22.Ph7 A1BNJ2 Costaria costata 23.Ph8 A1BNJ3 Ecklonia radiata 24.Ph9 A1BNJ4 Saccharina sessilis 25.Ph10 A1BNJ6 Macrocystis pyrifera Table-2 Physicochemical characterization of selected proteins Sr. No. Accession No. (Uniprot) Instability Index Aliphatic Index Grand Average of Hydropathicity (GRAVY) 1 Q09119 30.25 84.36 -0.171 2 Q760S7 32.25 85.39 -0.142 3 P24624 29.92 88.77 -0.116 4 P48691 25.50 83.98 -0.084 5 P23755 33.14 87.30 -0.195 6 Q760R5 30.95 85.39 -0.136 7 Q760T5 31.54 85.39 -0.142 8 Q20EX7 40.38 76.20 -0.330 9 Q0P3J3 35.54 79.12 -0.276 10 P12466 40.32 78.72 -0.301 11 P26958 39.52 79.54 -0.252 12 P08211 39.45 78.67 -0.247 13 Q2TGZ2 40.69 78.86 -0.254 14 B2X1Y2 40.82 79.94 -0.262 15 Q9T4F2 35.14 79.75 -0.243 16 A1BNI3 25.60 86.56 -0.081 17 A1BNI4 28.38 86.97 -0.089 18 A1BNI5 28.90 86.76 -0.094 19 A1BNI7 28.60 87.38 -0.089 20 A1BNI9 28.96 86.76 -0.095 21 A1BNJ1 28.96 86.76 -0.095 22 A1BNJ2 26.48 86.17 -0.094 23 A1BNJ3 27.11 86.97 -0.087 24 A1BNJ4 28.54 86.97 -0.092 25 A1BNJ6 28.37 87.38 -0.077 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(4), 11-17, April (2014) Int. Res. J. Biological Sci. International Science Congress Association 14 Table-3 Amino acid composition of rbcL proteins from various classes of algaeSr. No. Accession No. Amino Acid (%) A R N D C Q E G H I L K M F P S T W Y V O U 1 P23755 10.1 5.7 4.5 5.5 0.6 3.2 6.3 8.1 2.0 5.7 9.1 5.5 3.4 3.9 3.7 3.9 6.5 1.6 4.1 6.7 - - Figure-2 Homology modelling of Lessonia vadosa rubisco (viewed using Jmol) (PMDBID: PM0078256) Number of residues in favoured region (~98.0% expected): 456 (96.8%), Number of residues in allowed region (~2.0% expected): 12 (2.5%), Number of residues in outlier region: 3 (0.6%) Figure-3A Ramchandran Plot analysis using RAMPAGE International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(4), 11-17, April (2014) Int. Res. J. Biological Sci. International Science Congress Association 15 94.1% residues in favourable regions; 4.9% residues in additional residue regions; 0.7% residues in generously regions; 0.2% residues in disallowed regions; Over all G-factor: -0.01Figure-3B Ramchandran Plot analysis using PROCHECK Figure-3C Verify-3D plot for validity check of the modeled structure Figure-3D Errat Plot for validity check of the modeled structure International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(4), 11-17, April (2014) Int. Res. J. Biological Sci. International Science Congress Association 16 Functional characterization: CYS_REC (http://sunl.softberry.com/berry.phtml/topic) was used to locate “SS bond” between the pair of cystein residues, if present. The tool yields position of cysteins, total number of cysteins present and pattern, if present, of pairs in the protein sequence as output. The sequences for the chosen structures were only chosen for the determination of this parameter. The results are presented in table-4. Secondary structure content prediction: Secondary structure elements prediction was performed using the Secondary Structural Content Prediction (SSCP) server14. The results are shown in table-4. Table-4 Functional characterization and secondary structure content prediction Accession Number Cys_Rec Score Alpha helix Beta turns Coils P23755 No S-S bonds Negative values 43.4 17.0 39.5 P08211 172-192 57.7 19.8 23.3 56.9 449-459 85.1 A1BNI9 No S-S bonds Negative values 51.7 8.5 39.9 Superimposition and pairwise structural alignment: The selected 3D structures of the protein of the Chlorophyte (1GK8) and Phaeophyte (PM0078256) groups are superimposed and aligned pairwise with help of SuperPose version 1.0 15, which is a protein superposition server using the rhodophyte protein (1IWA) as the template and the chlorophyte (1GK8) and phaeophyte (PM0078256) proteins as the targets . Root Mean Square Deviation (RMSD) values were calculated between the set of targets and template protein to see how much modeled protein deviates from the template protein structure. The results are shown in table-5. Table-5 Pairwise structural alignment among the members of different classes of algae Template Target (PDB/ PMDB ID) RMSD 1IWA Galdieria sp.) 1GK8 (Chlamydomonas sp.) 1.369 PM0078256 (Lessonia vadosa) 0.712 Results and Discussion Sequence analyses: The Phylogenetic analyses of rbcL proteins gives important clues as each clade of the Phylogenetic tree comprised of the members of the same group viz. Rhodophyta, Chlorophyta or Phaeophyta (figure-1). Similar results were also observed elsewhere16. The physicochemical analyses of the rbcL was carried out using the ProtParam server (table-2), according to which a protein whose instability index is larger than 40 may be unstable17. Hence, the rbcL proteins from members of Rhodophyta and Phaeophyta groups were highly stable whereas those from Chlorophyta group were unstable or near to unstable. The aliphatic index of the proteins was found to be between 76.2-88.77; the higher indices suggesting they may be stable at high temperatures18. The GRAVY values19 fall in range of -0.077 to -0.330; indicating that the proteins will interact favourably with water. Structural analyses: The 3D structures of the two organisms, one each from Rhodophyta (Galderia sp.) and Chlorophyta groups (Chlamydomonas sp.)were further used for structural analyses. As there was no availability of experimental structures for any member of the Phaeophyta group, the 3D structure of the rubisco from Lessonia vadosa, a representative of the Phaeophyta group was modeled using the EsyPred Server (figure-2). The validity of the model was checked using RAMPAGE, Procheck, WHATIF, Errat and Verify-3d servers. Ramachandran plot for the same has been illustrated in figure- 3A (RAMPAGE) and figure-3B (PROCHECK). Altogether more than 90% of the residues were found to be in favoured and allowed regions, which validate the quality of homology model suggesting the acceptability of the modeled structure. Ramachandran Z-score as inferred from the WHATIF Server is 0.457. The score expressing how well the backbone conformations of all residues correspond to the known allowed areas in the Ramachandran plot, is within expected ranges for well-refined structures. The modeled structures were also validated by other structure verification servers such as Verify 3D and Errat (figure-3C, figure-3D). Verify 3D assigned a 3D- 1D score of �0.2 for 90.51% of the residues, representing the acceptability of the model This implies that the model was compatible with its sequence. ERRAT showed overall quality factor of 80.00 for the model. The validated homology model was thus submitted to the Protein Model Database with the PMDBID: PM0078256. The pairwise structure alignment was carried out using the red algal Rubisco (Galderia sp.), known to be the most potent rubisco enzyme, as template with that of the 3D structures of Chlorophyte (Chlamydomonas sp.) and Phaeophyte (Lessonia vadosa) proteins. RMSD (Root Mean Square Deviation) values obtained were 1.369 and 0.712 respectively, suggesting a greater similarity of the Phaeophyte protein with the red algal protein (table-5). Also the evolutionary closeness of the red and brown algal proteins is evident from the phylogenetic sequence analyses (figure-1). The present study shows close correlation between the Rhodophyte and the Phaeophyte rubisco at the functional level due to similarity in adaptability of the enzyme, which is comparable with ecology of both the type of organisms. The members of the Rhodophyte and Phaeophyte groups thrive in the marine habitat in contrast to most of the members of the Chlorophyta surviving in the freshwater habitat. Rubisco is the predominant enzymatic mechanism in the biosphere by which algae and other photosynthetic organisms fix CO into organic biomass via the Calvin–Benson–Basham reductive pentose phosphate pathway. Rubisco being the chief enzyme, in International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(4), 11-17, April (2014) Int. Res. J. Biological Sci. International Science Congress Association 17 photosynthesis, is acted upon by all forces of evolution20. Hence, adaptation of the organisms at their respective ecological niche plays an important role in the stereochemistry and structural similarities between their rubisco enzymes as evident from the present study between the members of different classes of algae. Conclusion The relationship of rubisco among the major three classes of algae was undertaken at the functional level by selection of the protein sequences (rbcL) and protein structures. The in silicostudies indicated a close correlation between that of Rhodophyta and Phaeophyta at the sequence as well as the structural levels. This is very well indicated in the phylogenetic analysis as well as the structural alignment analysis. The members of Rhodophyta and Phaeophyta belong to the same clade, indicating phylogenetic relatedness. Further, relatedness of brown and red algal protein structure is also evident by pair wise structural alignment. These results suggest that Phaeophyta may have more efficient Rubisco than that of Chlorophyta. Acknowledgement The instrumentation and computation facility under CAS (Gujarat govt.) and BIT Virtual, GSBTM, DST (Gujarat govt.) scheme are gratefully acknowledged. Financial support to the first author in form of UGC Research Fellowship in Sciences for Meritorious Students is also highly acknowledged. References 1.Spreitzer R.J. and Salvucci M.E., Rubisco: structure, regulatory interactions, and possibilities for a better enzyme, Annu. Rev. 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