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  • br Material and methods br Results and discussion br


    Material and methods
    Results and discussion
    Conclusion The gene encoding the Bos taurus muscle enolase enzyme was successfully isolated and cloned in this study. Optimization of the cloning, gene SB408124 and purification was performed and protein elution at 95% purity was achieved. An alternative method has been proposed to eliminate impurities in the purification process, which plays an important role in the biochemical characterization of the enzyme. It has been achieved to eliminate the extra band formation which was a constant problem throughout this particular experimental study. For SB408124 the first time in the literature, kinetic parameters of Bos taurus enolase 3, which plays a role in conversion of 2PG to PEP in the glycolytic pathway, was characterized by this study. In order to apply in silico studies, it is necessary to define the 3D structure of the enzyme. For this purpose, a homology model was built and the accuracy of this model was tested by web based evaluation and analysis programs. Substrate 2PG was docked into active site of the enzyme using molecular docking method for the analysis of interaction of enzyme-substrate complex. Enzyme-substrate complex built by homology modelling and molecular docking methods has been simulated by the molecular dynamics methods to stabilize structure and analyzed for its closeness to empirical results. The position of substrate and the final conformation of the complex were compared with the experimentally determined same molecules and their accuracy was analyzed. Overall, the reliable 3-D structure of the BtEno3-2PG complex is defined without the need of X-ray crystallography a time-consuming and expensive method and enzyme-substrate interactions are revealed. These in vitro and in silico analyses of the enolase from Bos taurus would enlighten further new drug development studies to be used in the treatment of theileriosis.
    Acknowledgements This research has been supported by Yildiz Technical University Scientific Research Projects Coordination Department. Project Number: 2015-07-04-KAP07. The numerical calculations reported in this paper were fully/partially performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources).
    Introduction Sunflower (Helianthus annuus L.) heterotrophic seeds, photosynthetically inactive during their development, rely completely on the maternal supply from photosynthetic tissues for building up reserves. Photosynthetic sucrose is exported to sink tissues providing the majority of carbon in seed lipids [1], [2]. Accordingly, sucrose is the principal source of carbon provided by the mother plant to the developing embryos [3]. Sucrose is enzymatically cleavage and hexoses and hexoses-phosphates enter the glycolysis pathway generating carbon, reducing equivalents, and energy for de novo intraplastidial fatty acid synthesis [4], [5]. Measurement of soluble carbohydrate levels in sunflower seeds have shown high hexose consumption associated with the synthesis of storage products, such as lipids [6]. Hexoses fate is mainly the cytosolic and plastidial glycolysis, both pathways interconnected by membrane transporters [7]. This transport of metabolites is fundamental for sunflower plastids due to previously described incapacity to use light energy [8]. In sunflower seed metabolism glucose-6 phosphate (G6P) has been identified like a major connexion between both subcellular compartments. Within plastids G6P feeds the oxidative pentose phosphate pathway (OPPP), generating the NADPH required to the de novo fatty acid synthesis [8], [9]. Triose phosphates (TP) constitute another chiefly connexion between cytosolic and plastidial glycolysis, most of the carbon in fatty acids is derived from these glycolytic intermediates. Although TP are found in both locations the comparison between enzyme activities found in the cytosol and within plastids in developing sunflower embryos has allowed the identification of the cytosolic pathway as the predominant source of carbon for lipid biosynthesis [6].