The class III anaerobic ribonucleotide reductases (RNRs) studied to date couple the reduction of ribonucleotides to deoxynucleotides with the oxidation of formate to CO 2 . Here we report the cloning and heterologous expression of the Neisseria bacilliformis class III RNR and show that it can catalyze nucleotide reduction using the ubiquitous thioredoxin/thioredoxin reductase/NADPH system. We present a structural model based on a crystal structure of the homologous Thermotoga maritima class III RNR, showing its architecture and the position of conserved residues in the active site. Phylogenetic studies suggest that this form of class III RNR is present in bacteria and archaea that carry out diverse types of anaerobic metabolism.T he class III ribonucleotide reductases (RNRs) are glycyl radical enzymes present in many strict and facultative anaerobes that catalyze the conversion of nucleotides to deoxynucleotides (1, 2) via a mechanism involving complex free radical chemistry and are largely responsible for providing the balanced pool of deoxynucleotides required for DNA synthesis and repair (3). The class III RNRs that have been characterized thus far obtain the reducing equivalents required to make deoxynucleoside triphosphates (dNTPs) from the oxidation of formate to CO 2 (4). Here we report a second subtype of class III RNR from Neisseria bacilliformis, which can obtain its reducing equivalents from the thioredoxin (TrxA)/thioredoxin reductase (TrxB)/ NADPH system. RNRs provide the only pathway for de novo biosynthesis of dNTPs (5). They share a structurally homologous active site architecture in the α subunit and a partially conserved, radicalbased reduction mechanism. RNRs have been isolated and characterized from all kingdoms of life and, based on the characterization of these proteins thus far, are divided into three classes (I, II, and III) according to the metallo-cofactor used to generate a thiyl radical that initiates the radical-dependent reduction chemistry (6). The class I RNRs use cofactors generated by the reaction of reduced metals (Fe, Mn, and Fe/Mn) and O 2 and are present only in aerobic organisms. The class II RNRs use adenosylcobalamin in an O 2 -independent reaction and are present in both aerobes and anaerobes. The class III RNR uses an O 2 -sensitive glycyl radical (G•) (2) situated in the α protein (NrdD), which is generated by a separate activating enzyme (NrdG) via radical S-adenosylmethionine (SAM)-[4Fe4S] 1+ chemistry (7,8). The class III RNRs are only found in facultative and obligate anaerobes. A second distinction between the three classes has been the source of the reducing equivalents for nucleotide reduction. In the class I and II RNRs, they are provided by a redoxin (thioredoxin, glutaredoxin, or NrdH), which is rereduced by thioredoxin reductase and NADPH (9-11). In contrast, for the bacteriophage T4 (12), its Gram-negative host Escherichia coli (1), and the Gram-positive Lactococcus lactis (13), the only class III RNRs characterized in detail to date, nucleotide reduction i...
The gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under HO stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce -mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.
Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.
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