ИСТИНА

NAD+-dependent formate dehydrogenases (FDH, EC 1.2.1.2) can be found in various organisms and is of great scientific and practical interest. At present time our laboratory pays high attention to formate dehydrogenases from plants, as they have unique kinetic properties among FDHs from other sources. For the first time our laboratory achieved super expression of FDH genes from plants Arabidopsis thaliana (AthFDH) and soya Glycine max (SoyFDH) in active and soluble forms and the enzymes were obtained as homogenious preparations. Study of these enzymes showed, that SoyFDH has the lowest Michaelis constant values for both substrates, compared with FDHs from bacteria and yeast. Due to that fact this enzyme can be successfully used as a biocatalyst for NADH regeneration. One of the most considerable disadvantages of SoyFDH is its low thermal stability. That is why one of the most important goals of our laboratory in this direction was improvement of thermal stability of SoyFDH by means of protein engineering. Analysis of the structure of the ternary complex [SoyFDH-NAD+-N3-] shown presence of hydrophobic Phe290 residue on the surface of the enzyme globule in the region of the coenzyme-binding domain. Our laboratory has already obtained three mutant forms of this enzyme, with amino acid substitutions by the residues Asp, Asn and Ser. This work is a further investigation of the role the residue Phe290 plays in properties of SoyFDH. This work results in obtaining and purification of five new mutant enzymes with amino acid substitutions of Phe290 with residues of Ala, Tyr, Glu, Gln and Thr, and analysis of their properties – thermal stability and kinetic properties. In all cases we observed improvement of both stability and catalytic properties. The mutant SoyFDH with the substitution by Glu residue shows maximum thermal stability improvement.