The tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1: Purification and properties

Abstract

NAD-dependent formate dehydrogenase (FDH1) was isolated from the α-proteobacterium Methylobacterium extorquens AM1 under oxic conditions. The enzyme was found to be a heterodimer of two subunits (α1β1) of 107 and 61 kDa, respectively. The purified enzyme contained per mol enzyme ≈ 5 mol nonheme iron and acid-labile sulfur, 0.6 mol noncovalently bound FMN, and ≈ 1.8 mol tungsten. The genes encoding the two subunits of FDH1 were identified on the M. extorquens AM1 chromosome next to each other in the order fdh1B, fdh1A. Sequence comparisons revealed that the α-subunit harbours putative binding motifs for the molybdopterin cofactor and at least one iron-sulfur cluster. Sequence identity was highest to the catalytic subunits of the tungsten- and selenocysteine-containing formate dehydrogenases characterized from Eubacterium acidaminophilum and Moorella thermoacetica (Clostridium thermoaceticum). The β-subunit of FDH1 contains putative motifs for binding FMN and NAD, as well as an iron-sulfur cluster binding motif. The β-subunit appears to be a fusion protein with its N-terminal domain related to NuoE-like subunits and its C-terminal domain related to NuoF-like subunits of known NADH-ubiquinone oxidoreductases.