In silico Characterization and Homology Modeling of Cyanobacterial RuBisCo (LS) with Computational Tools and Bioinformatic Servers

Author Name: *S. Meenakshi, S. Srisudha
Author Email: microkshi82@gmail.com

Abstract

Photosynthetic prokaryotes and eukaryotes, namely phytoplankton, chromophytes and cyanobacteria use the Calvin – Benson – Bassham (CBB) pathway to metabolize CO2 [1]. The key enzyme of the CBB pathway is ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo). RuBisCo-based CO2 fixation occurs in all cyanobacteria through C3 intermediates. The RuBisCo plays a pivotal role in linking the inorganic (CO2) and the organic phases (biomass) of the global carbon cycle.It is a slow and confused catalyst, limiting productivity and resource (e.g., water, nutrients) use efficiency in many plants [2]. The aim of this study was to determine the physicochemical properties of cyanobacterial RuBisCo and to develop 3-dimensional models of the selected enzyme. The biocomputational analysis was performed in silico using web-based software and servers. The alignment of cyanobacterial enzymes with the other related sequences and secondary structure analysis revealed that there are conserved amino acid substitutions and polymorphisms in the RuBisCo sequence. The large subunit of the selected cyanobacterial RuBisCo has many phosphorylation sites; alpha helical in structure, high homology with other cyanobacterial sequences (99%) and has close relation in phylogenetic tree. Furthermore, some cyanobacterial strains seem to posses unique amino acid stretches that may modulate various aspects of catalysis and regulation. The physicochemical characteristics and the 3D models provide a framework for the purification and characterization of cyanobacterial RuBisCos.

Keywords

Ribulose-1,5-bisphosphate carboxylase/oxygenase, cyanobacteria, (LS) Large subunit,computational analysis, proteomic tools.

Introduction

D-ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo, EC4.1.1.39), the most abundant enzyme on earth catalyses the atmospheric CO2 uptake in plants. It catalyses the addition of CO2 to ribulose-1,5-bisphosphate (RuBP) and gives two molecules of 3-phosphoglycerate (3-PGA). This reaction is essential for the synthesis of sugars through the Calvin cycle. Thereby, the carboxylase activity of RuBisCo is an important process in photosynthesis [3]. However, RuBisCo also catalyses the addition of O2 to RuBP to produce one molecule of 3-PGA and one molecule of 2-phosphoglycolate [4]. These pathways are relatively smaller contributors to global carbon fixation than that catalyzed by RuBisCo. Since RuBisCo is an important component in cellular metabolism, the focus has been emphasized more in learning about the molecular basis of RuBisCo’s ability to distinguish between CO2 and O2. Different forms of RuBisCo are widely distributed among the Photosynthetic organisms, Eukaryotic algae, Autotrophic Proteobacteria and some Archeae.
The catalytic incorporation of CO2 into RuBP by RuBisCo is the first step in the production of carbohydrates by plants, which are used to build biomass and produce energy during growth and development. Despite the pivotal role of RuBisCo in linking the inorganic (CO2) and the organic phases (biomass) of the global carbon cycle, it is a slow and confused catalyst, limiting the productivity and the use of resources (e.g., water, nutrients) in many plants [2]. Understandably, RuBisCo has been studied intensively and is a prime target for genetic engineering to improve photosynthetic efficiency [6]. Many amino acid residues have been manipulated in both subunits to understand the structure-function relationship of RuBisCo. The active site residues of LS (Large Subunit) are highly conserved among more than thousand LS sequences [7]. In Plant, Algae, Cyanobacteria and phototrophic and chemoautotrophic proteobacteria, RuBisCo comprises large subunit (LSU) chains and small subunit (SSU) chains. The substrate binding sites are located in the large Subunit. Many archaeal RuBisCo enzymes are found in obligate anaerobes that have evolved in the absence of molecular oxygen. Interestingly, those archaeal RuBisCo enzymes that have been studied were shown to be extremely oxygen-sensitive. The availability of internet based tools provides an excellent opportunity to characterize the physicochemical properties of Cyanobacterial RuBisCo as well as their primary, secondary and three dimensional structural properties.

Conclusion

Cyanobacterial RuBisCo LSs were selected from NCBI database and characterized for their physico-chemical properties. Molecular weight, isoelectric point, aliphatic index, instability, number of negative residues both positive and negative, Grand Average Hydropathy (GRAVY) and disulfide bond were computed. Physicochemical parameters provided useful data for the purification of cyanobacterial RuBisCo. Secondary structure analysis revealed that the proteins are largely alpha helical; this was supported by the homology modeling of RuBisCo from Geitlerinema sp. PCC 8501, Trichodesmium erythraeum IMS101, Arthrospira platensis NIES-39 and Thermosynechococcus vulcanus. Even though LS shows moderate active site and other properties the subunit enhances the functional activity of LS. Putative phosphorylation site can act as an inducer to LS to enhance the activity. The percentage of homology is 85% of Cylindrospermopsis to Zea mays and Sorghum bicolor, 87% to Oryza sativa japonica group. The percent of cyanobacterial homology shows 99% of Cylindrospermopsis to Raphidiopsis brookii D9 and Lyngbya majuscula 3L. The predicted phylogenetic trees based on LS Raphidiopsis brookii D9 and Fischerella were found to exhibit high similarity than other strains. The 3D model of Geitlerinema sp. PCC 8501 RuBisCo suggests that the molecule contain more beta-sheet and alpha helix.

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