NrdH-redoxin of Mycobacterium tuberculosis and Corynebacterium glutamicum dimerizes at high protein concentration and exclusively receives electrons from thioredoxin reductase.
NrdH-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. They function as the electron donor for class Ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. We solved the x-ray structure of oxidized NrdH-redoxin from Corynebacterium glutamicum (Cg) at 1.5 Å resolution. Based on this monomeric structure, we built a homology model of NrdH-redoxin from Mycobacterium tuberculosis (Mt). Both NrdH-redoxins have a typical thioredoxin fold with the active site CXXC motif located at the N terminus of the first α-helix. With size exclusion chromatography and small angle x-ray scattering, we show that Mt_NrdH-redoxin is a monomer in solution that has the tendency to form a non-swapped dimer at high protein concentration. Further, Cg_NrdH-redoxin and Mt_NrdH-redoxin catalytically reduce a disulfide with a specificity constant 1.9 × 10(6) and 5.6 × 10(6) M(-1) min(-1), respectively. They use a thiol-disulfide exchange mechanism with an N-terminal cysteine pKa lower than 6.5 for nucleophilic attack, whereas the pKa of the C-terminal cysteine is ~10. They exclusively receive electrons from thioredoxin reductase (TrxR) and not from mycothiol, the low molecular weight thiol of actinomycetes. This specificity is shown in the structural model of the complex between NrdH-redoxin and TrxR, where the two surface-exposed phenylalanines of TrxR perfectly fit into the conserved hydrophobic pocket of the NrdH-redoxin. Moreover, nrdh gene deletion and disruption experiments seem to indicate that NrdH-redoxin is essential in C. glutamicum.
Department of Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Brussels Center for Redox Biology, 1050 Brussels, Belgium.
Koen
K
Van Laer
Department of Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Brussels Center for Redox Biology, 1050 Brussels, Belgium.
Aleksandra M
AM
Dziewulska
Department of Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium.
Marcus
M
Fislage
Department of Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Brussels Center for Redox Biology, 1050 Brussels, Belgium.
Khadija
K
Wahni
Department of Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Brussels Center for Redox Biology, 1050 Brussels, Belgium.
Abderahim
A
Hbeddou
Brussels Center for Redox Biology, 1050 Brussels, Belgium; de Duve Institute, Université Catholique de Louvain, 1200 Brussels, Belgium; Welbio, 1200 Brussels, Belgium.
Jean-Francois
JF
Collet
Department of Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium.
Wim
W
Versées
Department of Molecular Biology, Area of Microbiology, University of León, 24071 León, Spain.
Luis M
LM
Mateos
Department of Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Brussels Center for Redox Biology, 1050 Brussels, Belgium; Welbio, 1200 Brussels, Belgium.
Veronica
V
Tamu Dufe
Department of Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Brussels Center for Redox Biology, 1050 Brussels, Belgium. Electronic address: joris.messens@vib-vub.be.
Joris
J
Messens
D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology; D020134:Catalytic Domain; D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D018360:Crystallography, X-Ray / Q000379:methods; D003545:Cysteine / Q000737:chemistry / Q000494:pharmacology; D019281:Dimerization; D004583:Electrons; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012265:Ribonucleotides / Q000737:chemistry; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013499:Surface Properties; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry; D013879:Thioredoxins / Q000378:metabolism*; D014965:X-Rays
D000995:Antitubercular Agents; D029968:Escherichia coli Proteins; D006020:Glycopeptides; C107179:NrdH protein, E Coli; D012265:Ribonucleotides; C089265:mycothiol; D007294:Inositol; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine