NAD(H) Regeneration-Driven Cascade Reaction of Formate Dehydrogenase and Diaphorase

Abstract

In this study, an NAD(H)-dependent formate dehydrogenase (FDH) is coupled with a ferredoxin-linked diaphorase (FdDP) to validate the cascade reaction that driven by the NAD(H)-regeneration. Despite a two orders of magnitude difference in their turnover rates (Kcat), the FDH-FdDP cascade demonstrated an enhanced FDH activity, proven by a significant increase in the formate oxidation activity during the initial reaction. The formate oxidation yield in the cascade reaction was directly quantified using a simple colorimetric method involving diluted HCl and KMnO4, achieving a detection limit as low as 10 µM, which surpasses the sensitivity of previously reported colorimetric approaches. While this specific pairing may not represent the most optimal combination due to inherent limitations of FDH, the results highlight the strong potential of FdDP as a versatile and universal NAD(H) regenerating enzyme, capable of pairing with other variety of NAD(H)-dependent catalytic enzymes. Furthermore, with the emerging advancements in enzyme engineering, the FDH and FdDP coupling represents a promising model for future applications in multi-enzymatic biosynthesis, including the development of self-sustaining bioelectrocatalytic systems.