초록
<P>Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO<SUB>2</SUB> reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO<SUB>2</SUB>-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO<SUB>2</SUB> capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from <I>Rhodobacter capsulatus</I> for its specificity toward NAD<SUP>+</SUP> vs. NADP<SUP>+</SUP> reduction. Starting from the NAD<SUP>+</SUP>-specific wild-type <I>Rc</I>FDH, key residues were exchanged to enable NADP<SUP>+</SUP> binding on the basis of the NAD<SUP>+</SUP>-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys<SUP>157</SUP>) in the β-subunit of the enzyme is essential for the binding of NAD<SUP>+</SUP>. RcFDH variants that had Glu<SUP>259</SUP> exchanged for either a positively charged or uncharged amino acid had additional activity with NADP<SUP>+</SUP>. The FdsB<SUP>L279R</SUP> and FdsB<SUP>K276A</SUP> variants also showed activity with NADP<SUP>+</SUP>. Kinetic parameters for all the variants were determined and tested for activity in CO<SUB>2</SUB> reduction. The variants were able to reduce CO<SUB>2</SUB> using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH.</P>