ИСТИНА

NAD-dependant formate dehydrogenase (FDH, EC 1.2.1.2.) is found in a wide range of living organisms, from bacteria and yeast to plants and fungi. It catalyses the reaction of NADH regeneration and plays a significant role in vital functions, especially in methylotrophic microorganisms. There this enzyme catalyses the final step of the catabolism of C1-compounds. Thus FDH can supply the cell with energy in the form of NADH. In an attempt to adapt themselves to stress conditions many pathogenic bacteria, including Staphylococcus aureus, can form biofilms, which is the most wide-spread way of bacterial existence in nature. Literature data claim that in Staphylococcus aureus in the form of a biofilm the RNA, coding formate dehydrogenase (SauFDH), stands on the third place among other RNAs quantitatively, while in the form of plankton there is no such crucial domination. The level of expression of SauFDH in biofilms is 20-fold higher, than in the planktonic state. Apparently, increased expression of SauFDH in biofilms aids their high survivability. During the analysis of the genomes of different pathogenic organisms (Staphylococcus aureus, Mycobacterium avium subsp. paratuberculosis, Bordetella bronchiseptica, Legionella) we found genes, encoding FDH-homologous proteins. After thorough studying of the amino acid sequence alignment of these proteins, we discovered a surprising fact: SauFDH shows a very low level of homology with other known FDHs. It is worth noticing that FDH is quite a conservative protein and even between two FDHs from different species absolute homology is about 40-50 percent. Nevertheless a host of conservative amino acid residues, specific to FDHs, remains intact in SauFDH. Thorough investigation of SauFDH properties can clarify the catalytic specificities of other formate dehydrogenases. The gene, encoding SauFDH was successfully cloned and expressed in E. coli cells as active and soluble enzyme. The developed system allowed us to obtain sufficient amount of recombinant FDH (the yield exceeded 500 mg per liter of media) without unnecessary contact with the bacterium Staphylococcus aureus. We also developed a system of high efficient enzyme purification. The analysis of SauFDH showed, that this enzyme possesses notable thermal stability compared to formate dehydrogenases from other sources, and an outstanding catalytic constant. The essential disadvantage of this enzyme turned out to be its Michaelis constants, both for NAD+ and formate. For better understanding the relation between the properties of SauFDH and its structure we obtained the crystal of the apo-form of this enzyme, which was successfully determined at 1.8A resolution. The work is supported by Russian Science Foundation (grant 16-04-00043).