Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, 34210 Istanbul, Turkey; Gumushane University, Faculty of Engineering and Natural Sciences, Department of Genetics and Bioengineering, 29100 Gumushane, Turkey.
Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, 34210 Istanbul, Turkey.
Int J Biol Macromol. 2018 Dec;120(Pt B):2346-2353. doi: 10.1016/j.ijbiomac.2018.08.184. Epub 2018 Aug 30.
Tropical theileriosis caused by Theileria annulata obligate parasite that infect ruminant animals, including Bos taurus. The disease results massive economic losses in livestock production worldwide. Here we describe cloning, expression and both biochemical and structural characterization of beta enolase from Bos taurus in vitro and in silico. The interconversion of 2‑phosphoglycerate to phosphoenolpyruvate was catalyzed by enolase is a metalloenzyme in glycolytic pathway and gluconeogenesis. Enolase from Bos taurus was cloned, expressed and the protein was purified at 95% purity using cobalt column by affinity chromatography. The optimum enzymatic activity was calculated at pH 6.5. For the first time in the literature, the kinetic parameters of the enzyme, Vmax and Km, were measured as 0.1141 mM/min and 0.514 mM, respectively. Besides, Bos taurus enolase 3-dimensional structure was built by homology modelling to be used in silico analyses. The interactions of the enzyme-substrate complex were elucidated by molecular dynamics simulations for 100 ns. These interactions were found to be the same as experimentally determined interactions in yeast. These results would enable further structure based drug design studies with the biochemical characterization of the host organism Bos taurus enolase enzyme in vitro and the elucidation of behavior of enzyme-substrate complex in silico.
环形泰勒虫引起的热带泰勒虫病是一种专性寄生虫，感染反刍动物，包括牛。这种疾病导致全球畜牧业生产遭受巨大的经济损失。在这里，我们描述了牛β烯醇酶的体外克隆、表达、生化和结构特征。烯醇酶将 2-磷酸甘油酸转化为磷酸烯醇丙酮酸，是糖酵解途径和糖异生中的一种金属酶。通过亲和层析用钴柱从牛中克隆、表达并纯化该蛋白，纯度达到 95%。最适酶活性在 pH 6.5 时计算。该文献首次测量了酶的动力学参数 Vmax 和 Km，分别为 0.1141 mM/min 和 0.514 mM。此外，还通过同源建模构建了牛烯醇酶的三维结构，以便进行计算机分析。通过分子动力学模拟对酶-底物复合物进行了 100 ns 的相互作用研究。这些相互作用与酵母中实验确定的相互作用相同。这些结果将有助于进一步进行基于结构的药物设计研究，包括对宿主生物牛烯醇酶的生化特征进行体外研究，以及对酶-底物复合物的计算机模拟行为进行阐明。
