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J Biol Chem. 2017 Jun 16;292(24):10123-10130. doi: 10.1074/jbc.M117.787754. Epub 2017 Apr 14.

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

Author information

1
From the Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
2
Scuola Universitaria Superiore IUSS Pavia, Piazza della Vittoria 15, 27100 Pavia, Italy.
3
Faculty of Pharmacy, University of Medicine and Pharmacy, Ho Chi Minh City, 41 Dinh Tien Hoang St., Ben Nghe Ward, District 1, Ho Chi Minh City, Vietnam, and.
4
Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy.
5
From the Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands, m.w.fraaije@rug.nl.

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.

KEYWORDS:

Thermobifida fusca; actinobacteria; deazaflavin; flavin; flavoprotein; nicotinamide; oxidation-reduction (redox)

PMID:
28411200
PMCID:
PMC5473218
[Available on 2018-06-16]
DOI:
10.1074/jbc.M117.787754
[Indexed for MEDLINE]
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