ИСТИНА

In plants NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is universal stress-induced enzyme. Plant FDHs are synthesized in cytoplasm and then transferred inside mitochondria due to specific signal peptide at N-terminus. In our laboratory we have created expression vectors to produce recombinant enzymes in E.coli cells using cDNA with genes of two FDHs from Arabidopsis thaliana (AraFDH) and soya Glycine max (SoyFDH). Optimization of expression resulted in yield of target enzyme up to 1 g per liter of cultivation medium. Thermal stability of AraFDH and SoyFDH were studied with kinetics of inactivation thermal and with differential scanning calorimetry. It was found that SoyFDH shows low thermal stability, while stability of AraFDH was similar to ones for FDHs from bacterium Mycobacterium vaccae N10 and yeast Candida boidinii. Plant FDHs showed the lowest Km values with formate and coenzyme between all studied FDH but catalytic constant was only 2.8-3.6 s-1 compared to 10 s-1 for FDH from bacterium Pseudomonas sp.101. SoyFDH showed the best values of catalytic efficiency (ratio kcat/Km) with formate and catalytic efficiency with NAD+ comparable with mutant PseFDH. Crystallization experiments were carried out in earth and space and crystal structures of apo- and holo-forms of AraFDH and SoyFDH were solved with resolution till 1.3Å. These structures were used for protein engineering experiments of SoyFDH. More than 20 single-point mutants were prepared and characterized. It was shown that only one residue is responsible for big differences in thermal stability between AraFDH and SoyFDH. About half of new SoyFDH mutants showed increase in catalytic parameters as well as in thermal stability. Combination of best amino acid changes in triple mutants provided further increase of thermal stability but catalytic parameters were the same as for wild-type enzyme