A Highly Active Formate Dehydrogenase Fdh3 toward NADP+ and Its Diaphorase Subunit from Hyperthermophilic Archaeon Thermococcus onnurineus NA1

Abstract

It has been identified that the genome of T. onnurineus NA1 possesses three genes, fdh1, fdh2, and fdh3, which were annotated as formate dehydrogenases (FDHs). Fdh2 forms a putative formate-dependent respiratory complex with Mfh2 hydrogenase and plays an essential role in formate-dependent energy metabolism. On the other hand, until recently, the functions and properties of Fdh1 and Fdh3 had not been revealed. In a recent study, we reported that the novel cytoplasmic NADP+-dependent Fdh3 consist of a tungsten-containing catalytic subunit (Fdh3A), a flavin-containing subunit (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). In contrast to the most NAD+-dependent FDHs, Fdh3 displayed a prominent preference for NADP+ as a cofactor. The catalytic efficiency (kcat/Km) of Fdh3 for NADP+ was measured to be 5,281 mM-1s-1, which is suitable for NADPH regeneration. In this study, we separately expressed and purified a putative NAD(P)+-reducing subunit Fdh3B from Escherichia coli Rosetta and T. onnurineus NA1, respectively. According to the structural modeling, Arg204 and Arg205 residues of Fdh3B may contribute to the stabilization of the 2-phosphate group of NADP(H). Fdh3B alone exhibited NADPH-diaphorase activity with a specific activity of 404 U/mg using benzyl viologen, which was determined at the optimal conditions of 70 ℃ and pH 9.5. These results suggest that the high NADP+ conversion rate of Fdh3 is derived from the properties of Fdh3B.