ИСТИНА

Formate dehydrogenase (FDH, EC 1.2.1.2.) plays a very important physiological role in different organisms and is a widely used enzyme for cofactor regeneration. FDH from bacterium Pseudomonas sp 101 (PseFDH) is one of the most efficient among other FDHs. Detailed studies of FDHs from different sources revealed special amino acid residues required for stability, specificity and activity. Objective: To obtain new mutants of PseFDH with improved stability and specificity to NADP+, which can be used as excellent biocatalysts for NADP+ regeneration. Methods: Search and selection of perspective mutations was done with rational design. New mutant enzymes were prepared by sequential site-directed mutagenesis. Mutant FDHs were overexpressed in E.coli, purified and characterized for kinetic parameters and stability (thermal inactivation kinetics study and differential scanning calorimetry). Results: We combined several approaches of genetic and protein engineering and obtained new FDHs with high thermal and operational stability and higher preference to NADP+. The base enzyme was mutant PseFDH SM4, obtained earlier in our lab. We added mutations, required for further thermal stabilisation (Ser141Ala, Ser160Ala - hydrophobization of α-helices), activity (mutation in “finger print” G(A)xGxxG region of FDH) and also a mutation required for to change coenzyme specificity form NAD+ to NADP+ (replacement of Asp221Gln). Substitution Cys145Ala resulted in improvement of chemical stability as well as KM for formate. As result we obtained several new mutants containing form 5 to 8 single amino acid changes. Thermal stability was improved by 1.2-3.0 fold and two of them were active with NADP+ then with NAD+. Conclusion: New mutants of PseFDH with higher thermal and operational stability and higher activity with NADP+ compared to previous variants were obtained. They can be effectively used as biocatalysts for NADP+ regeneration. The work was supported by the grant of the President of the Russian Federation for state support of young Russian scientists – Doctors of Science (contract – MD-2021) MD-349.2021.1.4