P72: Role of Ser156 residue in the cofactor specificity of Zymomonas mobilis ribitol dehydrogenase

Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to D-ribulose with concomitant reduction of NAD⁺ to NADH. ZmRDH is highly specific to NAD⁺ and virtually fails to catalyze the reaction with NADP⁺. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis of screened residues, was used to study the molecular determinants for the coenzyme specificity of ZmRDH. One homologous conserved amino acid, Ser156, in the substrate binding pocket of the wild-type ZmRDH was identified as an important residue affecting the coenzyme specificity of ZmRDH. Further insights into the function of residue Ser156 were obtained by substituting it with other non-polar hydrophobic or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered coenzyme specificity toward NAD⁺. In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP⁺ as cofactor. These data, isothermal titration calorimetric and molecular dynamics simulation studies suggest that Ser156 is critical for nicotinamide cofactor specificity of short-chain dehydrogenase/reductase (SDR) family.