ИСТИНА

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) was found in many different organisms: bacteria, fungi and plants. It plays a very important role in cell function. FDH is an enzyme of stress in plants. FDH is crucial enzyme for many pathogens to keep their life-sustaining activity in the form of biofilms. For example, the level of FDH mRNA in pathogenic bacterium Staphylococcus aureus (SauFDH) increases 20-fold when conditions change from plankton to biofilm and in last one it is in the third place compared to mRNA level of other enzymes in the cell. Multiple alignment of amino acid sequences of SauFDH and FDHs from many sources was done. It was found that the homology between of SauFDH and other FDHs is very low, but at the same time many of conservative residues, typical for all FDHs, are presented That is why we considered that SauFDH is of high scientific and practical interest. Recombinant FDHs from bacteria and yeasts are also widely used for coenzyme regeneration in processes of chiral and fine organic synthesis with NAD(P)+-dependent oxidoreductases. The main drawback of known FDHs is low specific activity (maximum 10 and 6.6 U/mg for bacterial and yeast enzymes, respectively). So, search of new FDHs with higher activity is very important. Gene of SauFDH was cloned and expressed in E.coli at the level up to 40-45% of total soluble cell proteins. Large scale cultivation was carried out and yield of the SauFDH was more than 500 mg per liter of the culture medium. High-efficient method of SauFDH purification was also developed and the enzyme was obtained in high amount. Properties of the recombinant SauFDH were studied and it was revealed, that thermal stability of the enzyme was one of the highest compared to described FDHs from other sources. Unexpected result was that the value of SauFDH specific activity was at least two-fold higher in comparison with one for bacterial FDHs. Unfortunately, Michaelis constants both, for NAD+ and formate, were higher compared to enzymes from other sources. Crystals of the apo-SauFDH were obtained and preliminary 3D structure was solved. The structure was deposited in PDB Data base as 5E0D. This work was supported by Russian Science Foundation (grant 16-14-00043).