Comparative 3D analysis was performed on the catalytic and the coenzyme-binding domains of NAD-dependent formate dehydrogenase (FDH). A structure of the Rossmann-fold type was revealed in the catalytic domain, and was assessed for homology with the canonical Rossmann folds in FDH and related enzymes. Both FDH domains were found to contain extensive regions comprising a Rossmann fold flanked by two alpha-helices and exhibiting high 3D homology. FDH proved to have an additional pseudosymmetry axis.

The comparative analysis of the primary and tertiary structures of NAD-dependent bacterial formate dehydrogenase (FDH) from methylotrophic bacterium Pseudomonas sp. 101 and a number of structurally characterized NAD-dependent dehydrogenases were performed. FDH has a highly conservative fold of the coenzyme binding domain. Position of the symmetry axis in the FDH molecule relative to the beta-sheets of its coenzyme binding domain with the respective sequences of the other NAD-dependent enzymes was performed on the basis of the spatial homology between these structures. Only one of the three amino acid residues previously thought to be conserved in the coenzyme binding domains of NAD-dependent dehydrogenases is preserved in the FDH molecule (Asp-221). Two glycine residues found in all previously studied dehydrogenases are substituted in FDH by Ala-198 and Pro-256, respectively. Position of the essential thiol of FDH (Cys-255) in the protein structure was established. It is suggested that Cys-255 is situated on or near polypeptide locus taking part in the conformational changes of the protein in the course of the catalysis.

Three heavy atom isomorphous derivatives were used for the X-ray analysis of the holo form of NAD-dependent bacterial formate dehydrogenase (ternary complex enzyme-NAD-azide) at 3.0 A resolution. The enzyme subunit contains a catalytic and a coenzyme binding domain, with the active centre and the coenzyme binding site in the cleft between the domains. The polypeptide chain's fold and the position of 393 C alpha-atoms were determined. The secondary structure of the formate dehydrogenase was resolved. The structure of the NAD-binding domain is shown to be similar to that of other NAD-dependent enzymes.

The primary structure of NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 is determined. The enzyme is composed of two identical subunits, each comprising 393 amino acid residues, and has a molecular weight of 43.1 kD. To elucidate the protein's amino acid sequence, four types of digestion were used: cyanogen bromide cleavage at methionine residues, endoproteinase Lys-C digestion at lysine residues, endoproteinase Glu-C cleavage at glutamic acid residues, and tryptic digestion. The peptides obtained were purified to homogeneity and characterized.