ИСТИНА

Rational design is widely used for production of enzymes with desired characteristics. In this work we report the change of the coenzyme specificity from NAD+ to NADP+ of formate dehydrogenase from bacterium Pseudomonas sp 101 (PseFDH) using this approach. Formate dehydrogenase (FDH, EC 1.2.1.2.) catalyses the reaction of formate oxidation coupled with reduction of NAD(P)+ to NAD(P)H. FDH is one of the best candidates for cofactor regeneration due to its wide range of pH optimum and practically irreversible reaction. One of the disadvantages of formate dehydrogenase is that the majority of these enzymes are more specific to NAD+ while NADPH regeneration is very important to lots of biotechnology processes. Genes from different bacteria, fungi and plants were successfully cloned and expressed in our laboratory. One of the most interesting FDHs is the enzyme from bacterium Pseudomonas sp 101 (PseFDH). It is the most stable among known native FDHs at high temperatures and has the best turnover number compared to FDHs from other sources. Analysis of 3D structure of PseFDH showed that one of the residues, responsible for coenzyme binding is Asp221. The substitution of this negatively charged residue to neutral amino acids might eliminate repulsion between carboxyl group of Asp221 and 2'-phosphate group of NADP+. Several new mutant forms with replacements of Asp221 were obtained and properties of the mutant enzymes were studied. It was shown that new mutants had a high specificity to NADP+. Also the thermal stability of some new mutant forms was studied by inactivation kinetics and differential scanning calorimetry (DSC).