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 RcFDH, 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.