Isolation and characterization of a thermostable F₄₂₀:NADPH oxidoreductase from Thermobifida fusca

Abstract

F₄₂₀H₂-dependent enzymes reduce a wide range of substrates that are otherwise recalcitrant to enzyme-catalyzed reduction, and their potential for applications in biocatalysis has attracted increasing attention. Thermobifida fusca is a moderately thermophilic bacterium and holds high biocatalytic potential as a source for several highly thermostable enzymes. We report here on the isolation and characterization of a thermostable F₄₂₀: NADPH oxidoreductase (Tfu-FNO) from T. fusca, the first F₄₂₀-dependent enzyme described from this bacterium. Tfu-FNO was heterologously expressed in Escherichia coli, yielding up to 200 mg of recombinant enzyme per liter of culture. We found that Tfu-FNO is highly thermostable, reaching its highest activity at 65 °C and that Tfu-FNO is likely to act in vivo as an F₄₂₀ reductase at the expense of NADPH, similar to its counterpart in Streptomyces griseus. We obtained the crystal structure of FNO in complex with NADP⁺ at 1.8 Å resolution, providing the first bacterial FNO structure. The overall architecture and NADP⁺-binding site of Tfu-FNO were highly similar to those of the Archaeoglobus fulgidus FNO (Af-FNO). The active site is located in a hydrophobic pocket between an N-terminal dinucleotide binding domain and a smaller C-terminal domain. Residues interacting with the 2′-phosphate of NADP⁺ were probed by targeted mutagenesis, indicating that Thr-28, Ser-50, Arg-51, and Arg-55 are important for discriminating between NADP⁺ and NAD⁺. Interestingly, a T28A mutant increased the kinetic efficiency >3-fold as compared with the wild-type enzyme when NADH is the substrate. The biochemical and structural data presented here provide crucial insights into the molecular recognition of the two cofactors, F₄₂₀ and NAD(P)H by FNO.