Atomic Resolution Crystal Structure of NAD(+)-Dependent Formate Dehydrogenase from Bacterium Moraxella sp. C-1

Abstract

The crystal structure of the ternary complex of NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Moraxella sp. C-1 with the cofactor (NAD+) and the inhibitor (azide ion) was established at 1.1 A resolution. The complex mimics the structure of the transition state of the enzymatic reaction. The structure was refined with anisotropic displacitalicents parameters for non-hydrogen atoms to a R factor of 13.4%. Most of the nitrogen, oxygen, and carbon atoms were distinguished based on the analysis of the titalicperature factors and electron density peaks, with the result that side-chain rotamers of histidine residues and most of asparagine and glutamine residues were unambiguously determined. A comparative analysis of the structure of the ternary complex determined at the atomic resolution and the structure of this complex at 1.95 A resolution was performed. In the atomic resolution structure, the covalent bonds in the nicotinamide group are somewhat changed in agreitalicent with the results of quantum mechanical calculations, providing evidence that the cofactor acquires a bipolar form in the transition state of the enzymatic reaction.

Fig. 1.

Photograph of crystals of the ternary MorFDH-NAD⁺-azide complex

Fig. 2.

Electron density map with 2|Fo| - |Fc| coefficients for residues Trp310, Gln313, and Pro314. The map is contoured at 1.0≤ (blue) and 3.5≤ (red) levels

Fig. 3.

Ribbon representation of the the ternary MorFDH-NAD⁺-azide complex. The subunits of the MorFDH dimer are related to each other by the crystallographic twofold rotation axis perpendicular to the plane of the figure. The NAD⁺ molecule is depicted in purple, the azide ion is depicted in red

Fig. 4.

Structure of the active site of MorFDH with the bound NAD⁺ molecule and azide ion a) Binding of the nicotinamide moiety of the NAD⁺ molecule and the azide ion by the active site residues (hydrogen bonds are depicted as dotted lines). b) Electron density map with 2|F0| - |Fc| coefficients countered at the 2.0≤ level for NAD⁺ and azide and the atomic numbering scheme for the nicotinamide moiety of NAD ⁺