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NAD+-dependent formate dehydrogenases(FDH) catalyze the oxidation of formate to CO2 with reduction of NAD+ to NADH. Eukaryotic FDHs catalyze this reaction by the ordered kinetic mechanism, whereas bacterial enzymes act by the random mechanism. The substrate channel in bacterial FDHs may be responsible for the random mechanism. Thus, search for substitutions blocking the substrate channel is helpful for better understanding of kinetic mechanisms at the molecular level. On the basis of a structural analysis and multiple alignment of FDHs representative sequences, we suggested following substitutions in FDH structure from Pseudomonas sp. (PDB ID 2NAC): F311W, Y102F and double F311W Y102F. All modifications were performed using the program UCSF Chimera. We used GROMACS software for their characterization. The F311W modification has demonstrated the minimum radius of the substrate channel (0,7 A after 6 ns of MD simulation). The superposition of 2NAC structure and Y102F demonstrates that hydrophobic side chains around substrate channel came closer together (per ~0,4 A) in Y102F after 30 ns of MD simulation. Resulting hydrophobic cavity hampers the charged substrate migration to active center, as it becomes energetically unfavorable. The structural analysis of double mutant F311W Y102F shows that the substrate channel is sterically blocked. Molecular modeling reveals the modifications blocking substrate channel of FDH (F311W Y102F, F311W, Y102F) which may be useful in following experimental investigation of FDHs kinetic mechanism. All simulations were performed using the facilities of the Supercomputer Centers “Lomonosov”, “Chebushev” [Sadovnichy et al., 2013] and “Arian Kuzmin”.