Formate dehyrogenase from Soya Glycine max: cloning, expression, properties and structure of the recombinant enzymeстатья

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[1] Formate dehyrogenase from soya glycine max: cloning, expression, properties and structure of the recombinant enzyme / A. A. Alekseeva, I. G. Shabalin, K. M. Polyakov, V. I. Tishkov // Journal of Biotechnology. — 2010. — Vol. 150, no. S1. — P. S476–S476. Formate dehydrogenases (FDHs, EC 1.2.1.2) form a group of enzymes found in both prokaryotes and eukaryotes that catalyse the oxidation of formate to carbon dioxide. Several years ago it was found, that FDH plays a very important role under stress conditions in plants, such as UV rays, high temperature, draught, etc. It is localized in mitochondria and the enzyme content can reach up to 9% of all mitochondrial proteins. Plant FDH precursor is expressed in cytoplasm followed by transport inside mitochondria due to presence of signal peptide in N-terminus. FDH from soya Glycine max (SoyFDH) was cloned and expressed in E.coli cells in active and soluble form. The enzyme was purified using hydrophobic chromatography on Phenyl Sepharose and gell-filtration through Sephadex S-200. The properties of the recombinant enzyme were studied and kinetic parameters (Km, Vm) were determined. It was found that SoyFDH has the lowest values of Michaelis constants for formate and NAD+ among all known formate dehydrogenases. Thermal stability of SoyFDH increases by the elevating of pH and ion strength. Inhibition constants of SoyFDH by several ions were determined. The method of determination of the enzyme active sites concentration was developed by measurement of fluorescence quenching during production of the ternary complex SoyFDH-NAD+-azide. Crystals of the enzyme ternary complex with NAD+ and azide were obtained and the X-ray structure of the complex was determined at 1.9Å resolution. [ DOI ]

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