A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon NA1.

The genome of the hyperthermophilic archaeon NA1 contains three copies of the formate dehydrogenase (FDH) gene, , , and . Previously, we reported that , clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was essential for formate-dependent growth with H production. However, the functionality of the other two FDH-coding genes has not yet been elucidated. Herein, we purified and characterized cytoplasmic Fdh3 to understand its functionality. The purified Fdh3 was identified to be composed of a tungsten-containing catalytic subunit (Fdh3A), an NAD(P)-binding protein (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). Fdh3 oxidized formate with specific activities of 241.7 U/mg and 77.4 U/mg using methyl viologen and NADP as electron acceptors, respectively. While most FDHs exhibited NAD-dependent formate oxidation activity, the Fdh3 of NA1 showed a strong preference for NADP over NAD as a cofactor. The catalytic efficiency ( ) of Fdh3 for NADP was measured to be 5,281 mM s, which is the highest among NADP-dependent FDHs known to date. Structural modeling suggested that Arg and Arg of Fdh3B may contribute to the stabilization of the 2'-phosphate of NADP(H). Fdh3 could also use ferredoxin as an electron acceptor to oxidize formate with a specific activity of 0.83 U/mg. Furthermore, Fdh3 showed CO reduction activity using reduced ferredoxin or NADPH as an electron donor with a specific activity of 0.73 U/mg and 1.0 U/mg, respectively. These results suggest a functional role of Fdh3 in disposing of reducing equivalents by mediating electron transfer between formate and NAD(P)H or ferredoxin.