FIGURE 2.

Specific activity of Ec.Mdh^5Q on OHB with NAD(P)H and 3D-protein structures with NAD⁺ or NADP⁺. (A) Catalytic efficiency (k_cat,app/K_M,app) of Ec.Mdh^5Q with NAD(P)H. Enzymatic activity was measured with purified enzyme at 37°C and pH 7.0 in 96-well flat-bottomed microtiter plates with 2 mM OHB and 0.25 mM NAD(P)H. The reactions were followed by monitoring the NAD(P)H absorption at 340 nm. Error bars indicate the standard deviation of the mean (n = 2). (B) X-ray structure of Ec.Mdh with bound NAD⁺ (Pdb code: 1emd). Side chains of amino acids in the selectivity control loop are shown (positions 34–38). (C) X-ray structure of Ec.Mdh with superimposed NADP⁺. The figure was generated by the structural alignment of Ec.Mdh with bound NAD (Pdb code: 1emd) and the malate dehydrogenase from Flaveria bidentis (Fb.Mdh) with bound NADP (Pdb code: 1civ). The structures of Fb-Mdh and NAD were omitted from the representation. (D) Model structure of Ec.Mdh D34G:I35S with superimposed NADP⁺. The figure was generated by structural alignment of the mutant enzyme model [generated with AlphaFold Colab (Jumper et al., 2021); mutations relative to wt enzyme are marked in violet] and the crystal structure of Fb.Mdh with bound NADP. Subfigures (B–D) were generated in PyMol v2.5.1 (http://www.pymol.org/pymol). The distance between selected residues is shown in Å. The carbon ribbon of cofactors NAD(P)⁺ is shown in pale yellow. The color scheme of NAD(P)⁺ and relevant amino acid side chains (position 34–38) is oxygen – red, nitrogen – blue, and sulfur – orange.