Abstract
The genome of the hyperthermophilic archaeon Thermococcus onnurineus NA1 contains three copies of the formate dehydrogenase (FDH) gene, fdh1, fdh2, and fdh3. Previously, we reported that fdh2, clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was essential for formate-dependent growth with H2 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 T. onnurineus NA1 showed a strong preference for NADP+ over NAD+ as a cofactor. The catalytic efficiency (kcat/Km) of Fdh3 for NADP+ was measured to be 5,281 mM−1 s−1, which is the highest among NADP-dependent FDHs known to date. Structural modeling suggested that Arg204 and Arg205 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 CO2 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.
Highlights
Formate dehydrogenase (FDH), a ubiquitous enzyme in prokaryotes and eukaryotes, catalyzes the reversible oxidation of formate to carbon dioxide (CO2)
The genome of T. onnurineus NA1 encodes three copies of genes annotated as putative formate dehydrogenase catalytic subunits, TON_0281, TON_1563, and TON_0539
Based on the multiple sequence alignment of various formate dehydrogenase (FDH), the catalytic residues of Fdh3 of T. onnurineus NA1 can be pinpointed as Cys133-His134-Arg324, which are well conserved in Escherichia coli, Rhodobacter capsulatus, and Desulfovibrio gigas (Figure 2A)
Summary
Formate dehydrogenase (FDH), a ubiquitous enzyme in prokaryotes and eukaryotes, catalyzes the reversible oxidation of formate to carbon dioxide (CO2). FDHs can be divided into two main classes according to their metal content/structure and. In contrast to metalindependent FDH, which mediates direct hydride transfer from formate to NAD+, in metal-containing FDH, the transfer of proton and electrons is mediated by the metal center in the active site and there is no direct proton/electron transfer between formate and the electron acceptor (Maia et al, 2015, 2017). The active site of FDHs is conserved and consists of three amino acids, Cys (or SeCys)-His-Arg. Many of them are known to be oxygenlabile, and FDHs containing tungsten or SeCys are generally more sensitive to oxygen (Nielsen et al, 2019). FDH of Methylobacterium extorquens AM1 containing tungsten was found to be oxygen tolerant (Laukel et al, 2003). The mechanism of oxygen-induced loss of activity has not yet been investigated in detail
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