ИСТИНА

NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) is an enzyme of high scientific and practical interest. FDH were found in bacteria, yeasts, fungi, moss, lichen and higher plants. Plant FDHs show lower values of Michaelis constants both for formate and NAD+ compared to ones from microorganisms. So, such FDHs can be successfully used for cofactor regeneration in fine organic synthesis especially for preparation of chiral compounds. Besides, in plants FDH plays a very important role in stress conditions. Its content dramatically increases during such stress conditions as drought, lack of oxygen, thermal discontinuity, pathogen infections, etc. Therefore, FDHs from plants are worth studying. The object of research in present work was formate dehydrogenase from soya Glycine max. The enzyme has the lowest values of Michaelis constants among even plant FDHs known at the moment. Rational design approach is successfully used in this laboratory to study structure-function relationship of different enzymes. Analysis of SoyFDH structure surface revealed the Phe residue in 290 position which is localized in coenzyme-binding domain of active site and its replacement may course changing in enzyme properties. Alignment of FDH amino acid sequences from different sources showed presence of residues Asn, Tyr, Asp, Ser. Computer modeling of influence of different amino acid changes of Phe290 was carried out and the most promising mutations were chosen for future work. Results of modeling shown that some of the Phe290 replacements may cause the formation of additional hydrogen bonds in the enzyme structure. Mutant SoyFDHs with changes of Phe 290 by Asn, Asp, Ser, Tyr, Gln, Glu, Thr and Ala were prepared and purified. Catalytic properties and stability of the mutant enzymes were studied. It was revealed, that practically all mutation influenced on Michaelis constant with formate, but KMNAD+ remained practically unchanged. Thermal stability of mutant SoyFDHs were studied by analysis of inactivation kinetics as well as by differential scanning calorimetry. It was found that mutations of Phe290 resulted also in significant increase of enzyme stability. One of the best enzymes SoyFDH Phe290Asp was 44 times more stable and had Tm 7.8oC higher, then that for wt-SoyFDH. This work was supported by Russian Foundation for Basic Research (grants 11-04-00920-а and 12-04-31740-mol a). Опубликовано FEBS Journal, 2013, v. 280, № S1, p.172-173