Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH...

Formate dehydrogenases are critical tools for nicotinamide cofactor regeneration, but their limited catalytic efficiency (k_cat/K_m) is a major drawback. A formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) was the first native NADP⁺-dependent formate dehydrogenase reported and has the highest k_cat/K_m toward NADP⁺ (k_cat/K_m^NADP+) compared with other FDHs that can utilize NADP⁺ as a hydrogen acceptor. However, the substrate and cofactor affinities of BstFDH are inferior to those of other FDHs, making its practical application difficult. Herein, we engineered recombinant BstFDH to enhance its HCOO^- and NADP⁺ affinities. Based on sequence information analysis and homologous modeling results, I124, G146, S262, and A287 were found to affect the binding affinity for HCOO^- and NADP⁺. By combining these mutations, we identified a BstFDH variant (G146M/A287G) that reduced K_m^NADP+ to 0.09 mM, with a concomitant decrease in K_m^HCOO-, and gave 1.6-fold higher k_cat/K_m^NADP+ than the wild type (WT). Furthermore, BstFDH I124V/G146H/A287G, with the lowest K_m^HCOO- of 8.51 mM, showed a catalytic efficiency that was 2.3-fold higher than that of the wild type and a decreased K_m^NADP+ of 0.11 mM. These results are beneficial for improving the performance of NADP⁺-dependent formate dehydrogenase in the NADPH regeneration of various bioreductive reactions and provide a useful guide for engineering of the substrate and cofactor affinity of other enzymes.