5N2I

F420:NADPH oxidoreductase from Thermobifida fusca with NADP+ bound

Summary for 5N2I

• Entry DOI: 10.2210/pdb5n2i/pdb
• Descriptor: Reduced coenzyme F420:NADP oxidoreductase, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, GLYCEROL, ... (4 entities in total)
• Functional Keywords: f420:nadph oxidoreductase, nadp+ bound, dimer of dimers, oxidoreductase
• Biological source: Thermobifida fusca (strain YX)
• Total number of polymer chains: 4
• Total formula weight: 100949.61

Authors

Kumar, H.,Nguyen, Q.-T.,Binda, C.,Mattevi, A.,Fraaije, M.W. (deposition date: 2017-02-07, release date: 2017-04-26, Last modification date: 2024-01-17)

Primary citation

Kumar, H.,Nguyen, Q.T.,Binda, C.,Mattevi, A.,Fraaije, M.W.
Isolation and characterization of a thermostable F420:NADPH oxidoreductase from Thermobifida fusca.
J. Biol. Chem., 292:10123-10130, 2017

PubMed Abstract: FH-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. 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 F: NADPH oxidoreductase (Tfu-FNO) from , the first F-dependent enzyme described from this bacterium. Tfu-FNO was heterologously expressed in , 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 as an F reductase at the expense of NADPH, similar to its counterpart in 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 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, F and NAD(P)H by FNO.

PubMed: 28411200
DOI: 10.1074/jbc.M117.787754

Experimental method

X-RAY DIFFRACTION (1.8 Å)