Staphylococcus aureusNrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase

Abstract

Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides, the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of deoxyribonucleotides during the cell cycle (29). Three main classes of RNRs are known. Class I RNRs are oxygen-dependent enzymes, class II RNRs are oxygen-independent enzymes, and class III RNRs are oxygen-sensitive enzymes. Class I RNRs are divided into two subclasses, subclasses Ia and Ib.

Implications of the Inability of Listeria monocytogenes EGD-e To Grow Anaerobically Due to a Deletion in the Class III NrdD Ribonucleotide Reductase for Its Use as a Model Laboratory Strain

“…In the presence of 1 to 2 mM dithiothreitol (DTT), TrxL significantly stimulated RNR activity (see Fig. S3 in the supplemental material), suggesting that in vivo it can function with thioredoxin reductase and NADPH as an electron donor for the aerobic class I RNR, in a manner similar to that of E. coli and S. aureus NrdH (39,45).…”

Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase

“…As a comparison, the K m of E. coli TR for its NrdH (CVQC active site) is 2.5-fold lower (1.1 M), and the V max is 89% that for E. coli Trx1 (28). The previously published data for the more closely related NrdH from S. aureus does not permit a direct comparison, but the activity appears to be low (51). (5), whereas NrdH redoxins are restricted to bacteria and seem to be exclusively involved in reduction of class Ib RNRs (12).…”

“…E. coli NrdH was found to give a K m of 0.3-0.6 or 1.2-1.6 M depending on whether the electrons were supplied by either DTT or TR/NADPH, whereas thioredoxin 1 was inactive (28). Aharonowitz and co-workers (51) found that both S. aureus NrdH and Trx1 were active as Table 1 and the E. coli proteins Trx1 and Trx2 plus Grx1-Grx4 were each subjected to -Blast, and the 200 top candidates after 6 -12 iterations were included in the comparison. In addition, all NrdH proteins listed in RNRdb were included prior to a ClustalX run with 1700 individual entries (after manual deletion of duplicates).…”

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.…”

“…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).…”