ИСТИНА

Formate dehydrogenase (EC 1.2.1.2, FDH) catalyzes the reaction of formate oxidation coupled with reduction of NAD(P)+ to NAD(P)H. The majority of FDHs has a high specificity to coenzyme NAD+ and doesn't catalyze the reaction with NADP+. There is know only one wild-type formate dehydrogenase from bacterium Burkholderia stabilis specific to NADP+. Nowadays NADPH is used in many important biotechnological processes. However, wild-type NADP+ specific FDH from B. stabilis is not suitable for practical application due to very high Km with formate. Therefore preparation of NADP+-specific FDH is actual and significant problem nowadays. Analysis of holo-structure of FDH from Pseudomonas sp. 101 (PDB2NAD) showed, that Asp221 residue plays an important role in coenzyme binding. Amino acid replacement of negatively charged Asp221 to none-charged residue may eliminate repulsion between carboxyl group of Asp221 and 2'-phosphate group of NADP+. That replacement would increase specific activity with NADP. In our laboratory the systematic study of formate dehydrogenases from different sources is carried out. One of the most interesting FDHs is formate dehydrogebnase from bacterium Pseudomonas sp 101. It has very high thermal stability and catalytic efficiency with NAD+. Previously many mutant forms of PseFDH with improved characteristics were obtained by genetic engineering. In this work new NADP+-dependent mutant form with substitutions in 221 positions are presented. The genes, encoding mutant enzymes were expressed in E.coli cells and new FDHs were obtained as active and soluble enzymes. Kinetic parameters in reaction with NAD+ and NADP+ were determined. It was shown that new mutant enzymes had a high specificity to NADP+. The double mutant PseFDH A198G/D221Q has the highest catalytic efficiency with NADP+ among all known NADP+-specific FDH. Study of its thermal stability of it was studied by both kinetic inactivation and differential scanning calorimetry (DSC). It was revealed, that it's more stable, than wt-PseFDH.