ИСТИНА

NAD+-dependent formate dehydrogenase (FDH, 1.2.1.2) is widely used for NAD(P)H regeneration in processes of fine organic and chiral synthesis with NAD(P)+-dependent oxidoreductases. Using rational design approach we prepared mutant formate dehydrogenases from bacteria1 and plants2,3. FDH from soya shows one of the best KM values for both substrates - formate and NAD+, but value of catalytic constant is about two times lower compared to one for FDH from methanol-utilizing bacteria (7 s-1). Unfortunately, catalytic constant of FDHs from methylotrophic bacteria is also low. We cloned and expresssed in E coli gene of FDH from pathogen bacterium Staphylococcus aureus (SauFDH). Level of expression of SauFDH was about 40% of cell soluble proteins. The enzyme was purified and characterized. It was found that specific activity of SauFDH was 2.5-fold higher compared to FDHs from methylotrophs. Thermal stability was studied through analysis of thermal inactivation kinetics at different temperatures and with differential scanning calorimetry. It was shown that only FDH from bacterium Pseudomonas sp.101 (PseFDH) has higher thermal stability than SauFDH. Native FDHs are NAD+-specific enzymes. There is only one exception4. We analyzed structure of PseFDH to change coenzyme specificity from NAD+ to NADP+. Only one mutation Asp221Gln resulted in preparation of NADP+-specific PseFDH. The second mutation Ala198Gly provided the highest catalytic efficiency with NADP+ compared to all described NADP+-specific FDHs. Additional ammino changes provided increase of thermal stability. This work was supported by Russian Science Foundation (project 16-04-00043)