Changing the Electron Acceptor Specificity of Rhodobacter capsulatus Formate Dehydrogenase from NAD + to NADP .

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