Abstract
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.
Highlights
NrdH-redoxins provide the electrons for the reduction of ribonucleotides
We investigate the NrdH-redoxins of C. glutamicum and M. tuberculosis
We found that the NrdH-redoxins receive electrons from thioredoxin reductase (TrxR) and not from MSH
Summary
NrdH-redoxins provide the electrons for the reduction of ribonucleotides. Results: We characterized NrdH-redoxin from Mycobacterium tuberculosis and Corynebacterium glutamicum. 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-. NrdH-redoxins are small glutaredoxin-like proteins often coded within the same operon as nrdE and nrdF, the two subunits of the class Ib RNR [12] They have a sequence identity in the range of 35– 85% with glutaredoxins (Grx) but receive electrons from thioredoxin reductase (TrxR) [10]. We identified mycoredoxins as a novel class of oxidoreductases in C. glutamicum and M. tuberculosis [16, 17] These enzymes use MSH to reduce disulfides in the cell and can be considered as the Grx analog of the actinomycetes. The pKa of the active site cysteines were determined, and the lack of nrdh gene deletion and disruption mutant in C. glutamicum showed that the protein might be essential for the cell
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