In Silico Studies of C3 Metabolic Pathway Proteins of Wheat (Triticum aestivum)

Photosynthesis is essential for plant productivity and critical for plant growth. More than 90% of plants have a C3 metabolic pathway primarily for carbon assimilation. Improving crop yields for food and fuel is a major challenge for plant biology. To enhance the production of wheat there is need to adopt the strategies that can create the change in plants at the molecular level. During the study we have employed computational bioinformatics and interactomics analysis of C3 metabolic pathway proteins in wheat. The three-dimensional protein modeling provided insight into molecular mechanism and enhanced understanding of physiological processes and biological systems. Therefore in our study, initially we constructed models for nine proteins involving C3 metabolic pathway, as these are not determined through wet lab experiment (NMR, X-ray Crystallography) and not available in RCSB Protein Data Bank and UniProt KB. On the basis of docking interaction analysis, we proposed the schematic diagram of C3 metabolic pathway. Accordingly, there also exist vice versa interactions between 3PGK and Rbcl. Future site and directed mutagenesis experiments in C3 plants could be designed on the basis of our findings to confirm the predicted protein interactions.


Introduction
Photosynthesis is arguably the most important energy conversion process on earth because the chemical energy it yields is the base of food chains that sustain the overwhelming majority of other life forms. Plants utilize atmospheric CO 2 to liberate oxygen and synthesize carbohydrates during photosynthesis. It is an event where radiant energy of sunlight is utilized to convert carbon dioxide into photosynthetic byproducts. On the basis of 1st product of photosynthesis or modi�cations in Calvin-Benson Cycle plants are grouped into three categories: C 3 plants, C 4 plants, and CAM plants. More than 90% of plants have C 3 metabolic pathway primarily for the carbon assimilation, whereas only 3% plants utilized C 4 metabolic pathway [1], and crassulacean acid metabolism (CAM) is found in sixteen thousand species of plants [2]. C 3 plants are less photosynthetically efficient and cannot grow in hot areas because of the activity of RuBisCO enzyme.
RuBisCo has the ability to �x both carbon dioxide and oxygen that result in the loss of carbon, nitrogen, and energy from plants [3]. Bread wheat (Triticum aestivum L.), durum wheat (Triticum turgidum L.), and barley (Hordeum vulgare L.) are the most outstanding C 3 crops in terms of cultivated area and food source, from the Holocene time up to present [4]. Improving crop yields for food and fuel is a major challenge for plant biology which is necessary to meet the requirements of a rapidly increasing world population [5]. Wheat is a common staple cereal food crop in all parts of the world and contributes to 28% of the world's edible dry matter (DM) and up to 60% of the daily calorie intake in several developing countries [6].
To enhance the production of wheat necessitates adopting strategies that can create the change in plants at the molecular level through one of two approaches: wet lab or computational. e study utilized computational bioinformatics and interactomics analysis of C 3 metabolic pathway proteins in wheat. Structural bioinformatics is concerned with the prediction, analysis, and visualization of the 3D structure of proteins. Besides NMR and X-ray Crystallography that entail enormous experimental costs, time, and laborious procedure, another recent and attractive approach built for the analysis of protein structure has recently emerged in structural bioinformatics. ere are three approaches primarily utilized to predict 3D structures of studied proteins: (1) homology modeling or comparative modeling [7,8], (2) threading or fold recognition [9][10][11], and (3) ab-initio prediction [12][13][14][15]. Proteins interact with each other as well as with other macromolecules to accomplish their functions within cell; therefore protein-protein interactions are complex and play a crucial role in most biological processes to determine the actual functioning of protein. Wet lab approaches such as tandem affinity puri�cation mass spectrometry [16], yeast two hybrid [17,18], and some others are found to enable the mapping of complex protein interactions. It is difficult to take advantage of these experimental techniques due to the complexity of biological molecules compared to computational protein-protein docking that o�er more bene�ts [19]. More recently, a high-throughput docking approach for protein-protein interaction has been reported [20]. e aim of this study is to improve the understanding of C 3 metabolic pathway proteins through in silico analysis and to study protein interaction in wheat. Each protein have has an important role, either in CO 2 �xation to regenerate ribulose 1, 5-bisphosphate or to synthesize starch and sucrose. is study selected only nine of proteins involved in the C 3 Pathway. e nine selected protein structures have not yet been determined through wet lab experiment (NMR, X-ray Crystallography) nor available in RCSB Protein Data Bank and UniProt KB.

Materials and Methods
A �owchart representing the use of tools in a sequential manner for study of nine important proteins, involved in C 3 metabolic pathway of wheat, is given in Figure 1. Sequences of these proteins were retrieved from NCBI (National Center for Biotechnology Information) in FASTA format, and it was used as a query for further analysis [21].
In order to generate three-dimensional models homology, threading and ab-intio approach was applied. Homology Modeling. e modeling of the 3D structure of the proteins was executed by Swiss-Modeler (http://swissmodel.expasy.org/) program [22,23]. reading Approach. SAM-T08 (http://compbio.soe.ucsc .edu/SAM_T08/T08-query.html) [24]. SAM-T08 web-based program for the modeling of three-dimensional structures of all selected protein sequences was used. A�-Initio Prediction. Iterative reading Assem�ly Re�nement (I-TASSER) [25,26]  Protein-Protein Interaction. Protein-protein interaction is generated by the help of HEX 6.1. HEX 6.1 program is used to speed up docking estimations; the method provides a feasible orientation rapidly and precisely [29].

Retrieval of Target and Recognition of Template Protein.
For this study, selected proteins involved in C 3 metabolic pathway were retrieved from NCBI (having maximum residual length), and their protein sequences in FASTA were used for a query sequence similarity search. PSI-BLAST identi�ed the potential template proteins with their respective PDB-ID with maximum similarity and E value. e BLAST results of nine target proteins are summarized in Table 1. As per our �ndings Rbcl and ATP synthase template showed 100% similarity with target sequence. GAPN and F-base demonstrated similarity more than 90%. 3PGK and WAXY showed percent similarity more than 80%, and remaining 3 proteins demonstrated the sequence similarity from 70 to 48%. Aer the selection of potential templates 3D model is generated by the use of Swiss-modeler program. We

Discussion
Over 90% of plants have C 3 metabolic pathway. C 3 photosynthetic pathway present in major crops such as wheat, barley, and rice. Wheat is major staple food crop all over the world. Decrease in productivity of wheat due to C 3 metabolic pathway involved oxygenation reactions and results in loss of energy 25-30%. Improve the efficiency of C 3 metabolic pathway needs to know the functions of proteins involved in C 3 pathway. Consequently, it is very crucial to be familiar with the 3D structure of proteins which gave insight into molecular functioning, which enhance the better understanding of physiological processes and biological systems. erefore in our study, eight protein sequences were used and modeled via threading approach, as these proteins have >700 amino acids, and models are constructed by SAM-T08. For one protein, that is, Sbe I (830 aa in length), I-TASSER program generated full length model (Figure 8). Full length generated models are signi�cant to use for future studies. Signi�cance of our modeling studies for nine proteins was depicted by con�rming that there is no crystalline structure present in protein databank till date.
On the basis of docking result we proposed the schematic diagram of C 3 metabolic pathway ( Figure 11). As per our analysis, Rbcl showed strong interaction with 3PGK in C 3 metabolic pathway, as we obtained minimum docking value (Table 4), while Rbcl as receptor indicated that it has no interaction with GAPN, Fba, WAXY, and PRK.
Our docking results also described that 3PGK as a receptor demonstrated the maximum interaction value with Rbcl. It is predicted that 3PGK regenerate the Rubisco enzyme. 3PGK showed strong linkage with two proteins, that is, Fba and GAPN. 3PGK interaction with Fba leads the process to starch biosynthesis, while its interaction with GAPN produces sucrose, as �nal products. Previous in vitro study depicted 3PGK (EC 2.7.2.3) and GAPN (EC 1.2.1.13) association [30].
Glyceraldehyde-3-P dehydrogenase (GAPN) is essential for plant metabolism and development. GAPN depicted strong interaction with SPS II in our analysis. SPS II are  [31,32]. In vivo it is shown that phosphorylation of SPS II changes the activity of SPS II and carbon partitioning [33]. In this regard SPS II enzyme is very essential for carbon partitioning, and also SPS II synthesize sucrose essential for plant development and growth. Sucrose formation is positively correlated with the rate of photosynthesis. We found no interactions of GAPN with Rbcl, Fba, ATP A, WAXY, and PRK.
Our results in Table 4 demonstrated that Fba has strong linkage with Sbe I. Fba is involve in the synthesis of starch, therefore they interact with the Sbe I help in starch formation. WAXY also showed strong interaction with Sbe I. Previous studies described that in rice developing endosperm C53 fragment (WAXY gene) interacts with two ACGT elements G-box and Hex (BZIP protein or SbeI), and both genes coordinately are regulated [34].
Sbe I demonstrated in (Table 4) the minimum value with the PRK revealing the strong interaction comparison to other proteins. PRK docking results demonstrated that it has strong interaction with 3PGK. It depicted that PRK regenerates the phosphoglycerate kinase which is also reported in a previous study by Raines in 2011 [35]. It demonstrated that reduced activity of PRK protein was enough to slower the photosynthesis process and decrease the plant productivity [36].
Our results predict that Fba, GAPN, and PRK have least interaction with other proteins in C 3 metabolic pathway. Similar to our results previous studies also demonstrated that GAPN, Fba, and PRK had no impact on carbon assimilation [37,38].

Conclusion
(i) During the model study we have predicted the selected nine proteins structures of C 3 pathway using bioinformatics methods as these are not determined through wet lab experiment (NMR, X-ray Crystallography) and were not available in RCSB Protein Data Bank and UniProt KB.
(ii) According to generalized known metabolic pathway Rbcl interacts with 3PGK only. But our study showed that 3PGK also interact with Rbcl to complete the CO 2 �xation pathway. So there also exist vice versa interactions of 3PGK with Rbcl. (iii) In biochemical pathways PRK interacts with Rbcl to complete the cycle, but according to our predicted pathway interaction between PRK with Rbcl is mediated through 3PGK. (iv) Future site directed mutagenesis experiments in C 3 plants could be designed on the basis of our �ndings to con�rm the predicted protein interactions.