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

Abstract

BackgroundRedox 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 FindingsA 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/SignificanceWe 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.

An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation

“…S6). Since FMN–DI has a smaller K m of ∼20–40 μM for NADH , under the high NADH concentration of 1 mM, we assumed that the reaction rate (initial velocity, V ) is directly correlated to the concentration of the “FMN–DI (DH) complex” (FMN–DI (DH) catalyzes the dehydrogenation reaction), where V ∼ k*[FMN–DI] and k ∼ k cat . By fitting the dehydrogenase activities with the addition of free FMN molecules, we were able to estimate the binding of FMN to DI protein.…”

“…The procedures of the expression and purification of a thermophilic DI has been described elsewhere . Briefly, a 636‐bp DNA fragment encoding DI (GenBank accession number JQ040550) was expressed in E. coli BL21(DE3).…”

“…The procedure was similar to what we published previously . Briefly, all solutions were prepared in an argon‐protected pyramid glove box.…”

Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways

“…NROR was also much more stable at temperatures than DI above 60 8C. [16,17] As shown in Ta ble 3, the best EM is BV, which has as pecific activity from NADPH to oxidized EM that is nearly 150 times higher than that of MV.A lthoughM V + is the beste lectrond onor for H 2 generation, its highly negative midpoint potential( À446 mV) resultsi nm uch slower rates for MV 2 + reduction by NROR.B yc onsidering data from Tables 2 and 3, the best EM to build ab iomimetic ETC (Figure 1 C)w as BV.…”

“…Reduced MV was the best mediator,e xhibiting nearly 100-fold higher H 2 production rates relative to that of NADPH. ReducedB Vw as not as good as reduced MV,p resumably due to its less negative reduction po- [16] and NAD(P)H:r ubredoxin oxidoreductase (NROR)f rom P. furiosus. [17] to regenerate reduced EM from NADPH.…”