ИСТИНА

NAD+-Dependent formate dehydrogenase (EC 1.2.1.2, FDH) widely occurred in nature. In plants the enzyme is localized in mitochondria. Plant FDHs are synthesized in cytoplasm as pro-enzyme which has signal peptide for transport inside mitochondria. Under stress conditions the content of FDH can achieve up to 8% of total mitochondria proteins. We have constructed plasmid with gene of FDH from soya G.max (SoyFDH) without signal peptide and SoyFDH was expressed in E.coli as soluble and active enzyme at the level up to 40% of cell soluble proteins and yield more that 0.5 g/L of cultivation medium. The enzyme was purified as homogeneous protein. It was shown that SoyFDH has the best KM values with NAD+ and formate compared to other known NAD+-dependent FDHs. At the same time the thermal stability of SoyFDH was significantly lower. Crystals of ternary complex [SoyFDH-NAD+-azide] were prepared and the structure was solved with resolution 1.9 Å. Structure analysis revealed potential sites for directed mutagenesis and about 20 mutant SoyFDHs were prepared and characterized. The best amino acid changes were combined in double and triple mutants. Thermal stability of the best mutant SoyFDH exceeded ones for all known wild-type FDHS except the enzyme from Pseudomonas sp.101. Specific activity was increased by 20-70% without change of KM values. Use of mutant SoyFDH for coenzyme regeneration in chiral synthesis with dehydrogenases is discussed. This work was supported by Russian Foundation for Basic Research (grant 11-04-00920) and Ministry of Education and Sciences of Russian Federation (contract 16.512.11.2148)