A Hidden Transhydrogen Activity of a FMN-Bound Diaphorase under Anaerobic Conditions

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Background Redox cofactors of NADH/NADPH participate in many cellular metabolic pathways for facilitating the electron transfer from one molecule to another in redox reactions. Transhydrogenase plays an important role in linking catabolism and anabolism, regulating the ratio of NADH/NADPH in cells. The cytoplasmic transhydrogenases could be useful to engineer synthetic biochemical pathways for the production of high-value chemicals and biofuels.

Methodology/Principal Findings A transhydrogenase activity was discovered for a FMN-bound diaphorase (DI) from Geobacillus stearothermophilus under anaerobic conditions. The DI-catalyzed hydride exchange were monitored and characterized between a NAD(P)H and a thio-modified NAD+ analogue. This new function of DI was demonstrated to transfer a hydride from NADPH to NAD+ that was consumed by NAD-specific lactate dehydrogenase and malic dehydrogenase.

Conclusions/Significance We discover a novel transhydrogenase activity of a FMN-DI by stabilizing the reduced state of FMNH2 under anaerobic conditions. FMN-DI was demonstrated to catalyze the hydride transfer between NADPH and NAD+. In the future, it may be possible to incorporate this FMN-DI into synthetic enzymatic pathways for balancing NADH generation and NADPH consumption for anaerobic production of biofuels and biochemicals.

proton-translocating transhydrogenase, escherichia-coli, nucleotide transhydrogenase, enzymatic pathway, nadh oxidase, purification, mechanism