Resolving Cys Residue Research Articles

Analysis and Characterization of Glutathione Peroxidases in an Environmental Microbiome and Isolated Bacterial Microorganisms.

Glutathione peroxidases (Gpx) are a group of antioxidant enzymes that protect cells or tissues against damage from reactive oxygen species (ROS). The Gpx proteins identified in mammals exhibit high catalytic activity toward glutathione (GSH). In contrast, a variety of non-mammalian Gpx proteins from diverse organisms, including fungi, plants, insects, and rodent parasites, show specificity for thioredoxin (TRX) rather than GSH and are designated as TRX-dependent peroxiredoxins. However, the study of the properties of Gpx in the environmental microbiome or isolated bacteria is limited. In this study, we analyzed the Gpx sequences, identified the characteristics of sequences and structures, and found that the environmental microbiome Gpx proteins should be classified as TRX-dependent, Gpx-like peroxiredoxins. This classification is based on the following three items of evidence: i) the conservation of the peroxidatic Cys residue; ii) the existence and conservation of the resolving Cys residue that forms the disulfide bond with the peroxidatic cysteine; and iii) the absence of dimeric and tetrameric interface domains. The conservation/divergence pattern of all known bacterial Gpx-like proteins in public databases shows that they share common characteristics with that from the environmental microbiome and are also TRX-dependent. Moreover, phylogenetic analysis shows that the bacterial Gpx-like proteins exhibit a star-like radiating phylogenetic structure forming a highly diverse genetic pool of TRX-dependent, Gpx-like peroxidases.

Basis for using thioredoxin as an electron donor by Schizosaccharomyces pombe Gpx1 and Tpx1

Glutathione (GSH) peroxidases (GPxs or GSHPx) and thioredoxin (Trx) peroxidases (TPxs) are two classes of peroxidases that catalyze the reduction of peroxides. GPxs and TPxs generally use GSH or Trx, respectively, to recycle the oxidized cysteine (Cys) residue in the protein. However, it is unclear why unlike human GPxs, the Schizosaccharomyces pombe Gpx1 (spGpx1) prefers Trx over GSH for recycling of the active-site peroxidatic Cys residue. Here, we compared spGpx1 and S. pombe Tpx1 (spTpx1) protein sequences with those of their respective homologs in Saccharomyces cerevisiae and humans. Our analysis revealed that like spTpx1, spGpx1 contains a pair of conserved Cys residues (Cys36 and Cys82). These two conserved Cys residues are named peroxidatic and resolving Cys residues, respectively, and are found only in GPxs and TPxs that prefer Trx as an electron donor. Our analysis suggested that Cys36 and Cys82 in spGpx1 are most likely to form a disulfide bond upon oxidation of Cys36. Molecular modelling predicted that a conformational change might be required for the formation of this disulfide bond. Evolutionary analysis suggested that fungal GPxs and TPxs are related by divergent evolution from a common ancestor. Our analyses support a prediction that while spGpx1 and spTpx1 are phylogenetically and functionally different, they evolved from a common ancestor and use a similar mechanism for recycling of the active-site peroxidatic Cys residue.

A novel mycothiol-dependent thiol\u2013disulfide reductase in Corynebacterium glutamicum involving oxidative stress resistance

ncgl2478 gene from Corynebacterium glutamicum encodes a thiol–disulfide oxidoreductase enzyme annotated as dithiol–disulfide isomerase DsbA. It preserves a Cys–Pro–Phe–Cys active-site motif, which is presumed to be an exclusive characteristic of the novel DsbA–mycoredoxin 1 (Mrx1) cluster. However, the real mode of action, the nature of the electron donor pathway and biological functions of NCgl2478 in C. glutamicum have remained enigmatic so far. Herein, we report that NCgl2478 plays an important role in stress resistance. Deletion of the ncgl2478 gene increases the size of growth inhibition zones. The ncgl2478 expression is induced in the stress-responsive extra-cytoplasmic function-sigma (ECF-σ) factor SigH-dependent manner by stress. It receives electrons preferentially from the mycothiol (MSH)/mycothione reductase (Mtr)/NADPH pathway. Further, NCgl2478 reduces S-mycothiolated mixed disulfides and intramolecular disulfides via a monothiol–disulfide and a dithiol–disulfide exchange mechanism, respectively. NCgl2478 lacks oxidase activity; kinetic properties of its demycothiolation are different from those of Mrx1. Site-directed mutagenesis confirms Cys24 is the resolving Cys residue, while Cys21 is the nucleophilic cysteine that is oxidized to a sulfenic acid and then forms an intramolecular disulfide bond with Cys24 or a mixed disulfide with MSH under oxidative stress. In conclusion, our study presents the first evidence that NCgl2478 protects against various stresses by acting as an MSH-dependent thiol–disulfide reductase, belonging to a novel DsbA–Mrx1 cluster.

Involvement of a mycothiol-dependent reductase NCgl0018 in oxidative stress response of Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial strain for amino acids and a key model organism for human pathogens. The study of C. glutamicum oxidoreductases, such as mycoredoxin 1 (Mrx1), dithiol-disulfide isomerase DsbA, and DsbA-like Mrx1, is helpful for understanding the survival, pathogenic infection, and stress resistance of its homologous species. However, the action mode and enzymatic function of C. glutamicum NCgl0018 preserving the Cys-Pro-Phe-Cys motif, annotated as a putative DsbA, have remained enigmatic. Here, we report that the NCgl0018-deleted strain increased sensitivity to various oxidative stresses. The ncgl0018 expression was induced in the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH- and organic peroxide- and antibiotic-sensing regulator (OasR)-dependent manner by stress. NCgl0018 reduced S-mycothiolated mixed disulfides and intramolecular disulfides via a monothiol-disulfide mechanism preferentially linking the mycothiol/mycothione reductase/NADPH electron pathway. Site-directed mutagenesis confirmed Cys107 was the resolving Cys residue, while Cys104 was the nucleophilic cysteine that was oxidized to a sulfenic acid and then could form an intramolecular disulfide bond with Cys107 or a mixed disulfide with mycothiol under stress. Biochemical analyses indicated that NCgl0018 lacked oxidase properties like the classical DsbA. Further, enzymatic rates and substrate preferences of NCgl0018 were highly similar to those of DsbA-like Mrx1. Collectively, our study presented the first evidence that NCgl0018 protected against stresses by functioning as a novel DsbA-like Mrx1 but not DsbA and Mrx1.

OsmC in Corynebacterium glutamicum was a thiol-dependent organic hydroperoxide reductase

Bacterial antioxidants play a vital role in the detoxification of exogenous peroxides. Several antioxidant defenses including low-molecular-weight thiols (LMWTs) and protective enzymes were developed to help the bacterium withstand the adverse stress. Although osmotically induced bacterial protein C (OsmC), classified as the organic hydroperoxide reductase (Ohr)/OsmC superfamily, has been demonstrated in some mycobacterial species, including M. tuberculosis and M. smegmatis, its physiological and biochemical functions in C. glutamicum remained elusive. Here we found the lack of C. glutamicum osmC gene resulted in decreased cell viability and increased intracellular reactive oxygen species accumulation under organic hydroperoxides (OHPs) stress conditions. The osmC expression was induced in the multiple antibiotic resistance regulator MarR-dependent manner by OHPs, and not by other oxidants or osmotic stress. Peroxide reductase activity showed that OsmC had a narrow range of substrates-only degrading OHPs, and detoxified OHPs mainly by linking the alkyl hydroperoxide reductase (AhpD) system (AhpD/dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide acyltransferase (SucB)). Site-directed mutagenesis confirmed Cys48 was the peroxidatic cysteine, while Cys114 was the resolving Cys residue that formed an intramolecular disulfide bond with oxidized Cys48. Therefore, C. glutamicum OsmC was a thiol-dependent OHP reductase and played important role of protection against OHPs together with Ohr.

Function of the evolutionarily conserved plant methionine-S-sulfoxide reductase without thecatalytic residue.

In plants, two types of methionine sulfoxide reductase (MSR) exist, namely methionine-S-sulfoxide reductase (MSRA) and methionine-R-sulfoxide reductase (MSRB). These enzymes catalyze the reduction of methionine sulfoxides (MetO) back to methionine (Met) by a catalytic cysteine (Cys) and one or two resolving Cys residues. Interestingly, a group of MSRA encoded by plant genomes does not have a catalytic residue. We asked that if this group of MSRA did not have any function (as fitness), why it was not lost during the evolutionary process. To challenge this question, we analyzed the gene family encoding MSRA in soybean (GmMSRAs). We found seven genes encoding GmMSRAs, which included three segmental duplicated pairs. Among them, a pair of duplicated genes, namely GmMSRA1 and GmMSRA6, was without a catalytic Cys residue. Pseudogenes were ruled out astheir transcripts were detected in various tissues and their Ka/Ks ratio indicated a negative selection pressure. In vivo analysis in Δ3MSR yeast strain indicated that the GmMSRA6 did not have activity toward MetO, contrasting to GmMSRA3 which had catalytic Cys and had activity. When exposed to H2O2-induced oxidative stress, GmMSRA6 did not confer any protection to the Δ3MSR yeast strain. Overexpression of GmMSRA6 in Arabidopsis thaliana did not alter the plant's phenotype under physiological conditions. However, the transgenic plants exhibited slightly higher sensitivity toward salinity-induced stress. Taken together, this data suggested that the plant MSRAs withoutthe catalytic Cys are not enzymatically active and their existence may be explained by a role in regulating plant MSR activity via dominant-negative substrate competition mechanism.

A thioredoxin-dependent peroxiredoxin Q from Corynebacterium glutamicum plays an important role in defense against oxidative stress.

Peroxiredoxin Q (PrxQ) that belonged to the cysteine-based peroxidases has long been identified in numerous bacteria, but the information on the physiological and biochemical functions of PrxQ remain largely lacking in Corynebacterium glutamicum. To better systematically understand PrxQ, we reported that PrxQ from model and important industrial organism C. glutamicum, encoded by the gene ncgl2403 annotated as a putative PrxQ, played important roles in adverse stress resistance. The lack of C. glutamicum prxQ gene resulted in enhanced cell sensitivity, increased ROS accumulation, and elevated protein carbonylation levels under adverse stress conditions. Accordingly, PrxQ-mediated resistance to adverse stresses mainly relied on the degradation of ROS. The physiological roles of PrxQ in resistance to adverse stresses were corroborated by its induced expression under adverse stresses, regulated directly by the stress-responsive ECF-sigma factor SigH. Through catalytical kinetic activity, heterodimer formation, and bacterial two-hybrid analysis, we proved that C. glutamicum PrxQ catalytically eliminated peroxides by exclusively receiving electrons from thioredoxin (Trx)/thioredoxin reductase (TrxR) system and had a broad range of oxidizing substrates, but a better efficiency for peroxynitrite and cumene hydroperoxide (CHP). Site-directed mutagenesis confirmed that the conserved Cys49 and Cys54 are the peroxide oxidation site and the resolving Cys residue, respectively. It was also discovered that C. glutamicum PrxQ mainly existed in monomer whether under its native state or functional state. Based on these results, a catalytic model of PrxQ is being proposed. Moreover, our result that C. glutamicum PrxQ can prevent the damaging effects of adverse stresses by acting as thioredoxin-dependent monomeric peroxidase could be further applied to improve the survival ability and robustness of the important bacterium during fermentation process.

Corynebacterium glutamicum methionine sulfoxide reductase A uses both mycoredoxin and thioredoxin for regeneration and oxidative stress resistance.

Oxidation of methionine leads to the formation of the S and R diastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown in Actinomycetes. Here, we report that a Corynebacterium glutamicum methionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of the msrA gene in C. glutamicum resulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of an S-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under stress conditions in vivo, the Trx/TrxR pathway alone is sufficient to reduce CgMsrA under normal conditions. Based on these results, a catalytic model for the reduction of CgMsrA by Mrx1 and Trx is proposed.

Mechanism of 1-Cys type methionine sulfoxide reductase A regeneration by glutaredoxin

Glutaredoxin (Grx), a major redox regulator, can act as a reductant of methionine sulfoxide reductase A (MsrA). However, the biochemical mechanisms involved in MsrA activity regeneration by Grx remain largely unknown. In this study, we investigated the regeneration mechanism of 1-Cys type Clostridium oremlandii MsrA (cMsrA) lacking a resolving Cys residue in a Grx-dependent assay. Kinetic analysis showed that cMsrA could be reduced by both monothiol and dithiol Grxs as efficiently as by in vitro reductant dithiothreitol. Our data revealed that the catalytic Cys sulfenic acid intermediate is not glutathionylated in the presence of the substrate, and that Grx instead directly formed a complex with cMsrA. Mass spectrometry analysis identified a disulfide bond between the N-terminal catalytic Cys of the active site of Grx and the catalytic Cys of cMsrA. This mixed disulfide bond could be resolved by glutathione. Based on these findings, we propose a model for regeneration of 1-Cys type cMsrA by Grx that involves no glutathionylation on the catalytic Cys of cMsrA. This mechanism contrasts with that of the previously known 1-Cys type MsrB.

Interaction of adenanthin with glutathione and thiol enzymes: Selectivity for thioredoxin reductase and inhibition of peroxiredoxin recycling

The diterpenoid, adenanthin, represses tumor growth and prolongs survival in mouse promyelocytic leukemia models (Liu et al., Nat. Chem. Biol. 8, 486, 2012). It was proposed that this was done by inactivating peroxiredoxins (Prxs) 1 and 2 through the formation of an adduct specifically on the resolving Cys residue. We confirmed that adenanthin underwent Michael addition to isolated Prx2, thereby inhibiting oxidation to a disulfide-linked dimer. However, contrary to the original report, both the peroxidatic and the resolving Cys residues could be derivatized. Glutathione also formed an adenanthin adduct, reacting with a second-order rate constant of 25±5M–1s–1. With 50µM adenanthin, the peroxidatic and resolving Cys of Prx2 reacted with half-times of 7 and 40min, respectively, compared with 10min for GSH. When erythrocytes or Jurkat T cells were treated with adenanthin, we saw no evidence for a reaction with Prxs 1 or 2. Instead, adenanthin caused time- and concentration-dependent loss of GSH followed by dimerization of the Prxs. Prxs undergo continuous oxidation in cells and are normally recycled by thioredoxin reductase and thioredoxin. Our results indicate that Prx reduction was inhibited. We observed rapid inhibition of purified thioredoxin reductase (half-time 5min with 2µM adenanthin) and in cells, thioredoxin reductase was much more sensitive than GSH and loss of both preceded accumulation of oxidized Prxs. Thus, adenanthin is not a specific Prx inhibitor, and its reported antitumor and anti-inflammatory effects are more likely to involve more general inhibition of thioredoxin and/or glutathione redox pathways.

Selenium as an Electron Acceptor during the Catalytic Mechanism of Thioredoxin Reductase

Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redox-active tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions (i) by being a better nucleophile than Cys, (ii) by being a better electrophile than Cys, (iii) by being a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys497-Sec498 dyad of the C-terminal redox center was mutated to either a Ser497-Cys498 dyad or a Cys497-Ser498 dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser497-Cys498 dyad formed a complex, and this structure has been determined by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat. Commun. 2, 383]. This experimental observation most likely means that the selenolate is the nucleophile initially attacking the disulfide bond of Trx because a complex resulted only when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the eight-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could accelerate this exchange reaction by being a good leaving group (attack at the sulfur atom) or by being a good electrophile (attack at the selenium atom). Here we provide strong evidence that the selenium atom is attacked in this exchange step. This was shown by creating a mutant enzyme containing a Gly-Gly-Seccoo- motif that had 0.5% of the activity of the wild-type enzyme. This mutant lacks the adjacent, resolving Cys residue, which acts by attacking the mixed selenosulfide bond that occurs between the enzyme and substrate. A similar result was obtained when Sec was replaced with homocysteine. These results highlight the role of selenium as an electron acceptor in the catalytic mechanism of thioredoxin reductase as well as its established role as a donor of an electron to the substrate.

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Peroxiredoxins (Prxs) detoxify peroxides and modulate H2O2-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1-4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H2O2 to form Cys sulfinic acid (Cys-SO2H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H2O2 and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.

P.2.008 5XFAD mouse model of Alzheimer's disease: a dissociation between brain pathology and behavioural phenotype

The diterpenoid, adenanthin, represses tumor growth and prolongs survival in mouse promyelocytic leukemia models (Liu et al., Nat. Chem. Biol. 8, 486, 2012). It was proposed that this was done by inactivating peroxiredoxins (Prxs) 1 and 2 through the formation of an adduct specifically on the resolving Cys residue. We confirmed that adenanthin underwent Michael addition to isolated Prx2, thereby inhibiting oxidation to a disulfide-linked dimer. However, contrary to the original report, both the peroxidatic and the resolving Cys residues could be derivatized. Glutathione also formed an adenanthin adduct, reacting with a second-order rate constant of 25±5 M–1 s–1. With 50 µM adenanthin, the peroxidatic and resolving Cys of Prx2 reacted with half-times of 7 and 40 min, respectively, compared with 10 min for GSH. When erythrocytes or Jurkat T cells were treated with adenanthin, we saw no evidence for a reaction with Prxs 1 or 2. Instead, adenanthin caused time- and concentration-dependent loss of GSH followed by dimerization of the Prxs. Prxs undergo continuous oxidation in cells and are normally recycled by thioredoxin reductase and thioredoxin. Our results indicate that Prx reduction was inhibited. We observed rapid inhibition of purified thioredoxin reductase (half-time 5 min with 2 µM adenanthin) and in cells, thioredoxin reductase was much more sensitive than GSH and loss of both preceded accumulation of oxidized Prxs. Thus, adenanthin is not a specific Prx inhibitor, and its reported antitumor and anti-inflammatory effects are more likely to involve more general inhibition of thioredoxin and/or glutathione redox pathways.

Cloning and functional characterization of a typical 2-Cys peroxiredoxin from southern bluefin tuna (Thunnus maccoyii)

Peroxiredoxins (Prxs, EC: 1.11.1.15) are cysteine-dependent peroxidases proposed to function as antioxidant enzymes and also in H2O2-mediated cell signaling. They have been well characterized in yeast, mammals, protists and bacteria but not yet in fish. Here we describe the cloning and functional characterization of a Prx 2 cDNA from southern bluefin tuna (SBT, Thunnus maccoyii), an important aquaculture species in South Australia. The SBT Prx sequence was closely related (76-92% identical) to Prx 1 and 2 sequences from other fish and mammals and phylogenetic analyses showed that it was most likely a Prx 2. The deduced amino acid sequence contained the peroxidatic and resolving Cys residues characteristic of typical 2-Cys Prx proteins from all kingdoms of life. It also contained the GGLG motif associated with the sensitivity of eukaryotic typical 2-Cys Prx proteins to overoxidation and consequent inactivation by H2O2. When the SBT Prx 2 was expressed in E. coli, it showed thioredoxin (Trx)-dependent peroxidase activity with H2O2, cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH). The SBT Prx displayed Michaelis-Menten kinetics with Trx but sigmoidal kinetics with H2O2 and CuOOH. The K(m)(Trx) was 12 microM and the S(0.5) values for H2O2 and CuOOH were 29 and 25 microM, respectively. At mM concentrations of H2O2, SBT Prx progressively lost its peroxidase activity as has been observed for other eukaryotic typical 2-Cys Prx proteins. The native SBT Prx enzyme existed as a mixture of dimers, tetramers, decamers and a higher order aggregate.

The selenoproteome of Clostridium sp. OhILAs: Characterization of anaerobic bacterial selenoprotein methionine sulfoxide reductase A

Selenocysteine (Sec) is incorporated into proteins in response to UGA codons. This residue is frequently found at the catalytic sites of oxidoreductases. In this study, we characterized the selenoproteome of an anaerobic bacterium, Clostridium sp. (also known as Alkaliphilus oremlandii) OhILA, and identified 13 selenoprotein genes, five of which have not been previously described. One of the detected selenoproteins was methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that repairs oxidatively damaged methionines in a stereospecific manner. To date, little is known about MsrA from anaerobes. We characterized this selenoprotein MsrA which had a single Sec residue at the catalytic site but no cysteine (Cys) residues in the protein sequence. Its SECIS (Sec insertion sequence) element did not resemble those in Escherichia coli. Although with low translational efficiency, the expression of the Clostridium selenoprotein msrA gene in E. coli could be demonstrated by (75)Se metabolic labeling, immunoblot analyses, and enzyme assays, indicating that its SECIS element was recognized by the E. coli Sec insertion machinery. We found that the Sec-containing MsrA exhibited at least a 20-fold higher activity than its Cys mutant form, indicating a critical role of Sec in the catalytic activity of the enzyme. Furthermore, our data revealed that the Clostridium MsrA was inefficiently reducible by thioredoxin, which is a typical reducing agent for MsrA, suggesting the use of alternative electron donors in this anaerobic bacterium that directly act on the selenenic acid intermediate and do not require resolving Cys residues.

Thioredoxin as a reducing agent for mammalian methionine sulfoxide reductases B lacking resolving cysteine

Previous reports described thioredoxin (Trx) as a very poor reductant for mammalian MsrB2 and MsrB3, which lack a resolving Cys residue. In contrast, we here report that Trx could reduce both MsrB2 and MsrB3 enzymes, similarly to the reduction of mammalian MsrA. We demonstrated that functional Trx is required for the reduction of these enzymes. We further identified MsrB2- or MsrB3-Trx complexes formed through intermolecular disulfide bonds involving catalytic residue of Trx. The present study provides evidence that the sulfenic acid intermediate of oxidized MsrBs lacking resolving Cys could interact with Trx and be directly reduced by this protein.