ИСТИНА

Formate dehydrogenase (FDH, EC 1.2.1.2.) catalyses a reaction of formate ion oxidation coupled with reduction of NAD+ to NADH. The enzyme is a widely spread in nature. Analysis of FDH sequences showed several common features in primary structure of the enzymes. For example, catalytically important Gln residue (Tishkov, V. et al. FEBS Lett. 1996) is placed in conserved motive – XPQP, where X in most bacterial enzymes is Phe (>90%), while in plants X is presented by Asn, Asp, Phe, and Tyr. In this work we studied role of residues in position X in three formate dehydrogenases from moss Physcomitrella patens (PpaFDH, X - Asn), bacterium Staphylococcus aureus (SauFDH, X - Tyr) and yeast Ogataea parapolymorpha (OpaFDH, X - Tyr). The next replacements were introduced in the FDHs - Y/A, Y/F and Y/Q in SauFDH, Y/D, Y/E, Y/A, Y/S in OpaFDH and N/D, N/E in PpaFDH. Data obtained showed that the residue X in XPQP motive plays a very important role in catalysis and protein stability in all studied FDHs. The best results for enzyme stability showed the substitutions of X to negatively charged residues Asp (PpaFDH and OpaFDH) and Glu (OpaFDH). In the case of SauFDH the replacements Y/A and Y/F practically did not changed the enzyme properties, but substitution Y/Q resulted in high increase of Km for both substrates - NAD+ and formate, and decrease of enzyme stability. The first Pro residue in motive XPQP in some very rare cases is substituted by another residue For exapmle, PpaFDH and FDH from yeast Saccharomyces cerevisiae (SceFDH) instead of Pro in the this position have Ala and Lys residues, respectively. The changes Ala/Pro in PpaFDH and Lys/Pro in SceFDH resulted in high increase of stability (about 4-10 times compared to wild type enzyme), but at the same time Km for NAD+ and formate (PpaFDH) or only Km for formate (SceFDH) also increased. This work was supported by Russian Science Foundation (grant 16-14-00043) and Russian Foundation for Basic Research (grant 17-04-01469) FEBS Journal, 2017, v.284, n S1, p.213.