Characterization of Escherichia coli NrdH

Abstract

Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductases. Either thioredoxin or glutaredoxin is a required electron donor for class I and II enzymes. Glutaredoxins are reduced by glutathione, thioredoxins by thioredoxin reductase. Recently, a glutaredoxin-like protein, NrdH, was isolated as the functional electron donor for a NrdEF ribonucleotide reductase, a class Ib enzyme, from Lactococcus lactis. The absence of glutathione in this bacterium raised the question of the identity of the intracellular reductant for NrdH. Homologues of NrdH are present in the genomes of Escherichia coli and Salmonella typhimurium, upstream of the genes for the poorly transcribed nrdEF, separated from it by an open reading frame (nrdI) coding for a protein of unknown function. Overexpression of E. coli NrdH protein shows that it is a functional hydrogen donor with higher specificity for the class Ib (NrdEF) than for the class Ia (NrdAB) ribonucleotide reductase. Furthermore, this glutaredoxin-like enzyme is reduced by thioredoxin reductase and not by glutathione. We suggest that several uncharacterized glutaredoxin-like proteins present in the genomes of organisms lacking GSH, including archae, will also react with thioredoxin reductase and be related to the ancestors from which the GSH-dependent glutaredoxins have evolved by the acquisition of a GSH-binding site. We also show that NrdI, encoded by all nrdEF operons, has a stimulatory effect on ribonucleotide reduction.Ribonucleotides are reduced to deoxyribonucleotides by ribonucleotide reductases, which are radical containing enzymes that may be divided into three main classes (1, 2).The electrons for this reaction are supplied by small redoxactive proteins such as thioredoxin (Trx) 1 or glutaredoxin (Grx) in the case of class I and II ribonucleotide reductase (3, 4), whereas formate fulfills this function for the anaerobic class III enzyme (5). Thioredoxin and glutaredoxin both contain two redox-active cysteine thiols in their reduced form, which by dithiol-disulfide interchange reduce an acceptor disulfide in the active center of ribonucleotide reductase. The active site sequences of thioredoxins and glutaredoxins are conserved among species, being Cys-Gly-Pro-Cys for thioredoxin and CysPro-Tyr-Cys for glutaredoxin (3, 4). The disulfide in oxidized thioredoxin is regenerated to a dithiol by thioredoxin reductase (TR) and NADPH, whereas oxidized glutaredoxin is reduced by 2 mol of GSH with the formation of GSSG, which is reduced by glutathione reductase (GR) and NADPH. Escherichia coli contains three different glutaredoxins (called Grx1, -2, and -3 (6)), which, like all glutaredoxins from other species, show high activity as general GSH-disulfide oxidoreductases in a coupled system with GSH, NADPH, and glutathione reductase (7). Three-dimensional structures for thioredoxins (8) and glutaredoxins (9, 10) show that they have essentially completely unrelated amino acid sequences but a similar overall fold (often referred to as the thioredoxin fold), con...

The Manganese-containing Ribonucleotide Reductase ofCorynebacterium ammoniagenes Is a Class Ib Enzyme

“…The NrdH product has been found to be a specific electron donor for the class Ib enzyme of S. typhimurium and L. lactis (12,37). The deduced NrdI product comprises 144 amino acid residues and has a predicted molecular mass of 15.8 kDa.…”

“…The hypothetical product encoded by the nrdH gene (75 residues, 8.3 kDa) corresponds to the previously described NrdH-redoxin from E. coli (37). The NrdH product has been found to be a specific electron donor for the class Ib enzyme of S. typhimurium and L. lactis (12,37).…”

Streptococcus sanguinis Class Ib Ribonucleotide Reductase

“…1) (2, 55). A number of artificial and endogenous systems are capable of mediating this re-reduction step, including dithiothreitol (DTT),Trx/TrxR/NADPH, Grx/ GSH/GR/NADPH, and NrdH/TrxR/NADPH, where Trx is thioredoxin, Grx is glutaredoxin, and GR is glutaredoxin reductase (6,11,20,21,56). The observation that nrdH is colocalized in the same operon with nrdE and nrdF in S. sanguinis and many other organisms makes this protein the most reasonable candidate for the endogenous reductant.…”

“…In many, but not all, of the class Ib RNRs, NrdH can re-reduce the disulfide in ␣ produced concomitant with dNDP formation (Fig. 1) and thus is required for multiple turnovers (6,11,(17)(18)(19). NrdH itself can be re-reduced in vitro by TrxR and NADPH in E. coli, B. anthracis, Staphylococcus aureus, and Corynebacterium glutamicum (6,11,20,21).…”

“…The subunits for these RNRs, ␣ and ␤, are designated NrdE and NrdF, respectively. Two additional gene products are often found within this operon as follows: NrdI, a flavodoxin that we have recently shown behaves as a flavin oxidase (10), and NrdH, a glutaredoxin-like thioredoxin (11,12). NrdF has been known for some time to be able to self-assemble an active Fe III 2 -Y ⅐ cofactor from apo-NrdF, Fe II , and O 2 in a manner analogous to the corresponding ␤ (NrdB) in the class Ia RNRs (13,14).…”