Graded Response of the Multifunctional 2-Cysteine Peroxiredoxin,CgPrx,to Increasing Levels of Hydrogen Peroxide in Corynebacterium glutamicum

Abstract

Aims:

Eukaryotic typical 2-cysteine (Cys) peroxiredoxins (Prxs) are multifunctional proteins subjected to complex regulation and play important roles in oxidative stress resistance, hydrogen peroxide (H₂O₂) signaling modulation, aging, and cancer, but the information on the biochemical functions and regulation mechanisms of prokaryotic atypical 2-Cys Prxs is largely lacking.

Results:

In this study, we show that at low peroxide concentrations, the atypical 2-Cys Prx in Corynebacterium glutamicum (CgPrx) mainly exists as monomers and displays thioredoxin (Trx)-dependent peroxidase activity. Moderate oxidative stress causes reversible S-mycothiolation of the H₂O₂-sensing Cys63 residue, which keeps CgPrx exclusively in dimer form with neither peroxidase nor chaperone activity. Then, the increased levels of H₂O₂ could act as a messenger to oxidize the redox-sensitive regulator hydrogen peroxide-inducible gene activator, leading to activation of expression of the more efficient mycothiol peroxidase and catalase to eliminate excessive peroxide. If oxidative stress is too severe, the H₂O₂-sensing Cys63 becomes hyperoxidized to sulfonic acid, which irreversibly inactivates the peroxidase activity, and most of CgPrx will be converted to multimeric chaperones for salvage of damaged proteins.

Innovation:

We demonstrate for the first time that atypical 2-Cys CgPrx acts as both a Trx-dependent peroxidase and a molecular chaperone and plays a regulatory role in modulating the peroxide-mediated signaling cascades.

Conclusion:

These results reveal that CgPrx functions as a multifunctional protein crucial for adapting appropriate responses to different levels of oxidative challenge in C. glutamicum. Antioxid. Redox Signal. 26, 1–14.

Introduction

Peroxiredoxins (Prxs), the predominant peroxidases for virtually all organisms, function to protect cells from oxidative stress by reducing hydrogen peroxide (H₂O₂), organic hydroperoxides, or peroxynitrite (19, 39). Differing from other peroxidases that require cofactors such as metal ions or prosthetic groups, Prxs use redox-active cysteines (Cyss) to reduce peroxides. Depending on the number of conserved Cys residues directly involved in catalysis, Prxs are grouped into three types: typical 2-Cys, atypical 2-Cys, and 1-Cys Prxs (15, 40). A strictly conserved and catalytically essential Cys called the peroxidatic cysteine (C_P) is shared within all types of Prxs. During the catalytic cycle, the C_P is first oxidized to sulfenic acid (C_P-OH); the sulfenic derivative is then stabilized by the formation of a disulfide bond with a resolving Cys (C_R) of another subunit (typical 2-Cys subfamily) or the same subunit (atypical 2-Cys subfamily). This disulfide bond is subsequently reduced by thioredoxin (Trx), using a direct thiol–disulfide exchange mechanism (15, 40). Unlike the 2-Cys Prxs, the sulfenic derivative of 1-Cys Prxs is reduced by thiol-containing electron donors such as ascorbate, low-molecular-weight thiols, or glutathione transferase π (28, 32, 37). The regenerated Prx is then ready for another catalytic cycle.

In addition to their peroxidase activity, Prxs have been reported to be involved in roles such as protecting DNA against mutation, suppressing tumor formation, promoting longevity, defending pathogens against host immune responses, acting as molecular chaperones to stabilize proteins, and regulating redox signal transduction (18, 24, 33, 34, 40, 50, 51). Under oxidative stress conditions, the peroxidatic Cys-SOH of 2-Cys Prxs is occasionally further oxidized to sulfinic or sulfonic derivatives, which have lost the peroxidase activity. Oxidization also triggers the structure of Prx to transit from low-molecular-weight (LMW) oligomers with peroxidase activity to high-molecular-weight (HMW) complexes with molecular chaperone activity (8, 18). The equilibrium of Prx oligomers is regulated by post-translational modifications and by cues such as redox state, ionic strength, and pH (5, 29, 30).

Innovation

In this study, we demonstrate that the atypical 2-cysteine (Cys) thioredoxin-dependent peroxidase 2-Cys peroxiredoxin in Corynebacterium glutamicum (CgPrx) also functions as a molecular chaperone. The switch between these two functions is regulated by oxidative stress-triggered S-mycothiolation, which is concomitant with significant changes in its protein structure. We also demonstrate that CgPrx plays a regulatory role in modulation of peroxide-mediated signaling cascades by controlling the activation of hydrogen peroxide (H₂O₂)-inducible gene activator and expression of catalase (katA) and mycothiol peroxidase (mpx). Our data establish that CgPrx, by acting as a delicate H₂O₂ sensor and the primary scavenger of H₂O₂, is central for making appropriate responses to different levels of H₂O₂ faced by C. glutamicum.

As a reversible post-translational thiol modification, glutathionylation has been reported to protect the active site Cys residues of certain Prxs from irreversible overoxidation to sulfonic acids, regulate the quaternary structure of some typical 2-Cys Prxs, or to provide reducing equivalents to support peroxidase activity (6, 25, 32, 36). Moreover, inactivation of eukaryotic 2-Cys Prx by hyperoxidation or phosphorylation has been proposed to promote the localized accumulation of H₂O₂ and thus regulate the activity of other proteins (10, 50, 51). However, most of the current knowledge on Prxs was obtained in eukaryotic cells; information about bacterial Prxs was largely lacking because of their robust response to oxidative inactivation and appeared to be required only to degrade peroxides.

Corynebacterium glutamicum, one of the most important industrial bacteria widely used for production of amino acids and nucleotides, is also an excellent laboratory model for systems biology study and for the investigation of its pathogenic relatives such as C. diphtheriae, C. jeikeium, and Mycobacterium tuberculosis (44). C. glutamicum exhibits remarkable resistance to H₂O₂, a feature that has drawn considerable attention for mechanistic studies (27). C. glutamicum produces mycothiol (MSH; AcCys-GlcN-Ins) as the main nonenzymatic antioxidant. MSH is functionally equivalent to glutathione (γ-l-glutamyl-l-cysteinylglycine) and is important in defense against diverse stresses, including oxidative agents, alkylating compounds, heavy metal ions, and antibiotics (23, 31). C. glutamicum also produces a large battery of antioxidant enzymes, such as catalase (katA) (46), mycothiol peroxidase (MPx) (38, 45), and mycoredoxin 1 (Mrx1) (35), to prevent reactive oxygen species (ROS)-induced cell damage.

Recently, Chi et al. found that S-mycothiolation of the atypical 2-Cys peroxidase in C. glutamicum (CgPrx) inhibits the peroxidase activity and that such inhibition can be reversed by reduction mediated by the Mrx1/MSH/mycothiol disulfide reductase electron pathway (7). While the dual functions and switching mechanisms between different functions and structures of typical 2-Cys Prxs have been well documented, the molecular chaperone activity has not yet been reported for any atypical 2-Cys Prxs. This study contributes to a deeper characterization of the atypical 2-Cys Prx, CgPrx, in C. glutamicum. A model in which CgPrx functions as a multifunctional protein crucial for adapting appropriate responses to different levels of oxidative challenge in C. glutamicum was proposed.

Results

CgPrx is required for resistance to low oxidative stress

Sequence analyses revealed that CgPrx belongs to the atypical 2-Cys Prx group, whose members have been shown to function as Trx-dependent monomeric peroxidases (20) (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/ars). Therefore, we speculated that CgPrx receives electrons from the CgTrx-reducing system (CgTrx/CgTrxR [thioredoxin reductase]) to reduce peroxides. As expected, CgTrx1 and CgTrx2, but not CgTrx3, supported the peroxidase activity of CgPrx in reducing H₂O₂ and cumene hydroperoxide (Fig. 1A and Supplementary Fig. S2). The rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption significantly reduced in the absence of the CgTrx system, further supporting that CgPrx functions as a Trx-dependent peroxidase (Fig. 1A). However, no NADPH consumption was observed by using the CgMrx1/CgMtr/MSH-reducing system (Supplementary Fig. S3), indicating that this system cannot provide electrons to CgPrx.

FIG. 1.

CgPrx was required for resistance to low concentrations of H₂O₂ in Corynebacterium glutamicum. (A) Trx-dependent peroxidase activity of CgPrx. Peroxidase activity was determined by recording NADPH oxidation at 340 nm in the presence of different Trx-reducing systems and shown with the progress curves. The reactions omitted CgTrx, CgPrx, or H₂O₂, which served as negative controls. Similar results were obtained in three independent experiments, and data shown are from one representative experiment done in triplicate. (B) Survival rates for WT(pXMJ19), Δprx(pXMJ19), Δprx(pXMJ19-prx), and WT(pXMJ19-prx) strains after being challenged with various concentrations of H₂O₂. Mean values with standard deviations (error bars) from at least three repeats are shown. *p ≤ 0.05. (C) Intracellular ROS levels in WT(pXMJ19), Δprx(pXMJ19), Δprx(pXMJ19-prx), and WT(pXMJ19-prx) strains were measured by H₂DCFDA fluorescence determination assay after exposure to indicated concentration of H₂O₂. Fluorescence intensity was expressed in AU. Mean values with standard deviations (error bars) from at least three repeats are shown. **p ≤ 0.01. (D) Protein carbonyl contents were analyzed by Western blotting with anti-dinitrophenyl antibody after exposure to indicated concentration of H₂O₂ at 30°C for 30 min (upper panel). A parallel run was stained with Coomassie Brilliant Blue (lower panel). AU, arbitrary unit; CgPrx, 2-Cys Prx in Corynebacterium glutamicum; Cys, cysteine; H₂DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; H₂O₂, hydrogen peroxide; NADPH, nicotinamide adenine dinucleotide phosphate; Prx, peroxiredoxin; ROS, reactive oxygen species; Trx, thioredoxin; WT, wild-type.

To investigate the physiological function of CgPrx, we tested the effects of deleting this gene on the sensitivity of C. glutamicum to H₂O₂. Compared with the wild type, the Δprx mutant was more sensitive to low concentrations of H₂O₂ (60 and 100 mM), and the sensitive phenotype can be almost completely complemented by expressing the prx gene (Fig. 1B). Interestingly, the mutant exhibited similar sensitivity to high concentrations of H₂O₂ (150 and 200 mM) to that of the wild type (Fig. 1B). In agreement with this observation, overexpression of CgPrx increased the resistance of the wild type to low, but not high, concentrations of H₂O₂ (Fig. 1B).

Since Prx is known to eliminate ROS under oxidative stress in various organisms (14, 21), we next examined the ability of CgPrx in ROS removal by fluorometrically measuring intracellular ROS levels with the membrane permeate fluorogenic probe, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) (48). Comparing with the wild type, the Δprx mutant contained significantly higher ROS after being exposed to low concentrations of H₂O₂ for 30 min. This higher ROS levels in the mutant can be reversed by expressing the prx gene (Fig. 1C). The higher level of ROS in the mutant was not detected when the cells were exposed to higher concentrations of H₂O₂ (Fig. 1C). To test whether CgPrx is capable of reducing oxidative damage to proteins under low H₂O₂ concentrations, we performed carbonyl assays with proteins isolated from the Δprx mutant and wild-type cells that had been treated with H₂O₂. A 30-min exposure to <100 mM H₂O₂ caused more carbonylation in proteins from the mutant (Fig. 1D). Such difference was not observed when cells were treated with 150 mM H₂O₂. Taken together, these results demonstrate that CgPrx plays an important role in protecting C. glutamicum cells against oxidative damage caused by low levels of H₂O₂.

Cys63 and Cys97 form an intramolecular disulfide bond during the reduction of H₂O₂

CgPrx contains three Cys residues in which Cys63 and Cys97 likely are the peroxidatic and the resolving Cyss (Supplementary Fig. S1), respectively, essential for the activity of atypical 2-Cys Prxs (51). In reaction, the peroxidatic Cys residue first is oxidized by peroxide to a Cys sulfenic acid (C_P-SOH) intermediate (12); this peroxidatic Cys-SOH is then attacked by the resolving Cys of Prx to form a redox-active disulfide. To stabilize and trap the Cys-SOH, the C-terminal Cys of the active site disulfide pair must be removed (12). To test this, we produced CgPrx variants, CgPrx:C84SC97S, CgPrx:C63SC97S, and CgPrx:C63SC84S, and treated the proteins with 4-chloro-7-nitrobenzofurazan (NBD-Cl) under anaerobic condition with or without prior exposure to H₂O₂. H₂O₂-treated CgPrx:C84SC97S labeled with NBD gave a maximal absorbance at 347 nm characteristic of the NBD-modified product [R-S(O)-NBD], indicating the detection and trapping of approximately stoichiometric amounts of SOH by Cys63, the only Cys in this variant (Fig. 2A). In contrast, CgPrx:C84SC97S treated with NBD-Cl, but not H₂O₂, gave a maximal absorbance at 420 nm, typical for the thiol adducts with NBD-Cl (Cys-S-NBD) (42). In contrast, the absorbance of the CgPrx:C63SC97S and CgPrx:C63SC84S variants remained unchanged upon incubation with NBD-Cl either before or after exposure to H₂O₂. These results further support that Cys63 is the peroxidatic Cys residue in CgPrx, a conclusion that was strengthened by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS/MS) analysis, which detected a peak of 2135.7 Da in the H₂O₂-treated CgPrx:C84SC97S (labeled [B] in inset panel), 16 Da higher compared with the untreated protein resulting from the addition of an oxygen atom and formation of Cys sulfenic acid (Cys-SOH) on the Cys63 residue (Fig. 2B and Supplementary Fig. S4A).

FIG. 2.

Redox response of CgPrx variants. (A) Formation of sulfenic acid on Cys-63 determined by the NBD-Cl assay. Spectrophotometric analysis of NBD-labeled CgPrx variants. Hollow circle, triangle, and rhombus expressed 10 mM H₂O₂-treated CgPrx:C84SC97S, CgPrx:C63SC84S, and CgPrx:C63SC97S proteins, respectively. Solid circle, triangle, and rhombus expressed H₂O₂-untreated CgPrx:C84SC97S, CgPrx:C63SC84S, and CgPrx:C63SC97S proteins, respectively. (B) Analysis of sulfenic acid formation in CgPrx by MALDI-TOF-TOF MS/MS. The H₂O₂-treated CgPrx band excised from nonreducing gel in inset panel (labeled [B]) was treated with trypsin and subjected to MS analysis. Only the relevant portion of the mass spectrum is shown. (C) Determination of Cys oxidation in CgPrx variants with the AMS assay. CgPrx variants (25 μM) treated with DTT (50 mM) were further incubated with or without 25 mM H₂O₂, and then free thiol groups were covalently modified with AMS. The modified samples were separated by 15% nonreducing SDS-PAGE. (D) Analysis of disulfide bond formation by MALDI-TOF-TOF MS/MS. Bands excised from nonreducing gels in (C) (labeled [D]) were treated with trypsin and subjected to MS analysis. Only the relevant portion of the mass spectrum is shown. AMS 4-acetamido-4′-maleimidyldystilbene-2, 2′-disulfonic acid; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; NBD-Cl, 4-chloro-7-nitrobenzofurazan; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

To investigate whether Cys97 acts as the resolving Cys residue of CgPrx, we analyzed the formation of disulfide bonds in single-Cys variants of CgPrx (CgPrx:C63S, CgPrx:C84S, and CgPrx:C97S) in response to H₂O₂ using the 4-acetamido-4′-maleimidyldystilbene-2, 2′-disulfonic acid (AMS) covalent modification assay. AMS covalently modifies free thiol groups, leading to slower electrophoretic mobility proportional to the number of free thiol groups in the protein (11). AMS-treated oxidized CgPrx:C84S migrated more rapidly compared with similarly treated reduced CgPrx:C84S, but similar to that of oxidized untreated CgPrx:C84S (Fig. 2C), indicating that CgPrx:C84S was fully oxidized by H₂O₂ to form a disulfide bond between Cys63 and Cys97. However, CgPrx:C97S, treated with H₂O₂ and modified with AMS, migrated faster than its reduced form (AMS modified) and slower than that of the oxidized (unmodified) form (Fig. 2C), indicating that despite modification, the protein was not fully oxidized. Oxidized and reduced CgPrx:C63S were modified by AMS and have the same electrophoretic mobility, indicating that these proteins were not oxidized by H₂O₂ and Cys84/Cys97 still existed in the thiol state. These indicate that CgPrx:C84S is fully oxidized by H₂O₂ to form a disulfide bond between Cys63 and Cys97. This conclusion was confirmed by MS analysis. The protein bands from H₂O₂-treated CgPrx:C84S (labeled [D] in Fig. 2C) have a mass of 3945.9 Da, which corresponds to the 50-to-69 peptide (calculated and observed mass, 2119.5 Da) and the 96-to-112 peptide (calculated and observed mass, 1828.2 Da) of the CgPrx:C84S variant connected by a disulfide bridge that does not appear in non-H₂O₂-treated CgPrx:C84S (Fig. 2D and Supplementary Fig. S4B). These results demonstrated that CgPrx is capable of forming a disulfide bond between the peroxidatic Cys63 and the resolving Cys97 during H₂O₂ decomposition.

CgPrx switches from a peroxidase to a molecular chaperone upon sulfonation of Cys63 by high concentrations of H₂O₂

Several typical 2-Cys Prxs have been reported to have dual physiological functions as peroxidase and molecular chaperone (1, 2, 8, 18). To investigate whether the atypical 2-Cys Prx CgPrx possesses molecular chaperone activity, we examined its ability to inhibit thermal aggregation of malate dehydrogenase (MDH) at high temperature. Incubation of more than 7.5 mM H₂O₂-treated CgPrx and MDH at a 1:1 subunit molar ratio prevented the latter from aggregation (Fig. 3A). Similarly, CgPrx treated with higher than 7.5 mM H₂O₂ effectively protected insulin from dithiothreitol (DTT)-induced protein precipitation (Fig. 3B), further suggesting that CgPrx acts as an effective molecular chaperone. Importantly, CgPrx not treated with H₂O₂ displayed high peroxidase activity, but not chaperone activity. The peroxidase and molecular chaperone activities of CgPrx were regulated by H₂O₂ (Fig. 3C). CgPrx exhibits high peroxidase activity in the presence of low concentrations of H₂O₂ (0.1 mM) for 30 min; its molecular chaperone activity is negligible under this condition. As the concentration of H₂O₂ increases, its peroxidase activity declines, accompanied by increased molecular chaperone activity. The chaperone activity of CgPrx peaks in the presence of 25 mM H₂O₂ and is about 2–2.5-fold weaker compared with the well-known molecular chaperone, heat shock protein 70 (Fig. 3C).

FIG. 3.

Protein structure-dependent switches of the peroxidase and chaperon activities of CgPrx in vitro . (A, B) Chaperone activities of CgPrx were determined by measuring the thermal aggregation of MDH at 43°C (A) or DTT-induced chemical aggregation of insulin at 25°C (B) at 1:1 molar ratios of CgPrx:substrate. BSA and HSP 70 were used as negative and positive control, respectively. Similar results were obtained in three independent experiments, and data shown are from one representative experiment done in triplicate. (C) Relative activities of the peroxidase (right Y axis, closed rhombus) and chaperone (left Y axis, open round) functions of CgPrxs pretreated with various concentrations of H₂O₂. The activities were compared with those of CgPrx with DTT pretreatment. (D, E) Structure changes of purified CgPrx protein (10 μM) treated with indicated concentrations of H₂O₂ for 30 min were analyzed by 15% native PAGE (D) and 15% nonreducing SDS-PAGE (E). MS result of band (labeled *) was shown in Supplementary Fig. S4C. (F) MALDI-TOF-TOF MS/MS analysis of the CgPrx tetramer. Bands (labeled [F]) excised from nonreducing gels in (E) were treated with trypsin and subjected to MS analysis. Only the relevant portion of mass spectrum is shown. The novel peak with m/z 2167.2 from a tryptic peptide was present. BSA, bovine serum albumin; HSP 70, heat shock protein 70; MDH, malate dehydrogenase.

Interestingly, H₂O₂-induced switch in the biochemical activity of CgPrx occurs concomitant with significant changes in its protein structure. H₂O₂-treated CgPrx migrated as multiple bands in both native polyacrylamide gel electrophoresis (PAGE) and nonreducing sodium dodecyl sulfate (SDS)-PAGE gels (Fig. 3D, E). The abundance of protein bands corresponding to oligomers increased as the concentration of H₂O₂ increases (Fig. 3D, E). In contrast, CgPrx not treated with H₂O₂ existed as a monomer (Fig. 3D, E).

CgPrx appeared to exist mainly as intramolecular disulfide bond-containing monomers when treated with low concentrations of H₂O₂ (0.1, 1, and 7.5 mM) (Fig. 3D, E). High concentrations of H₂O₂ (15 and 25 mM) induced the formation of multimers, including dimers, tetramers, and hexamers (Fig. 3D, E). A novel peak of 2167.2 Da peptide that was 48 Da higher than that of the Cys-63-containing 56–99 peptide (LVLNIFPSVDTGVCATSVRK) in reduced CgPrx was observed in tetrameric and monomeric CgPrx (labeled [F] and [*] in Fig. 3E), suggesting that the CgPrx was sulfonated on the Cys63 residue when treated with high concentrations of H₂O₂ (Fig. 3F and Supplementary Fig. S4C, D). Thus, CgPrx functions as a peroxidase in the form of monomers in low concentrations of H₂O₂ and switches to oligomers with chaperone activity upon being sulfonated on the Cys63 residue when treated with high concentrations of H₂O₂.

Oxidative stress-induced structural changes of CgPrx in vivo

To examine whether the structure of CgPrx is regulated by oxidative stress in vivo, we treated cells of the Δprx mutant overexpressing His₆-CgPrx with various concentrations of H₂O₂ and probed the forms of His₆-CgPrx by immunoblotting after nonreducing SDS-PAGE separation. Our results indicate that in the absence of oxidative stress, CgPrx existed as reduced monomers and became intramolecular disulfide bond-containing monomers when exposed to oxidative stress conditions (<120 mM H₂O₂) (Fig. 4A, B). One hundred twenty micromolar H₂O₂ induced most proteins into its dimer form (labeled [C] in Fig. 4A). Interestingly, an extra mass of 2605.2 Da was identified in this dimer, which was 484 Da higher than the Cys-63-containing (56–99) peptide of reduced CgPrx (Fig. 4C and Supplementary Fig. S4D). This observation is consistent with the result from the addition of MSH, indicating that Cys63 was mycothiolated under certain oxidative stress. This observation was confirmed by the reversible biotin switch assay. His₆-CgPrx overexpressed in the Δprx mutant pretreated with 120 mM H₂O₂ exhibited a strong band of mycothiolation. However, no mycothiolation signal was observed in the non-H₂O₂-treated Δprx(pXMJ19-His₆-prx) strain. Similarly, no mycothiolation signal was observed for His₆-CgPrx overexpressed in the MSH null mutant, ΔprxΔmshC, regardless of 120 mM H₂O₂ treatment (Fig. 4E).

FIG. 4.

Oxidative stress-dependent structural changes of CgPrx in vivo . (A) Proteins extracted from Δprx(pXMJ19-His₆-prx) cells exposed to indicated concentrations of H₂O₂ for 10 min were resolved on nonreducing SDS-PAGE and analyzed with Western blotting by using the anti-His antibody. In the last lane, the 150 mM H₂O₂-treated sample with an additional 120-min recovery in the absence of H₂O₂ is shown. (B–D) Analysis of disulfide bonds, S-mycothiolation, and sulfonic acid formed on CgPrx by MALDI-TOF-TOF MS/MS. Bands (labeled [B], [C], and [D]) excised from nonreducing gels in (A) were treated with trypsin and subjected to MS analysis. Only the relevant portion of each mass spectrum is shown. (E) Mycothiolation of CgPrx was monitored by the biotin switch assay. The IAM-alkylated protein extracts of Δprx(pXMJ19-His₆-prx) and ΔmshCΔprx(pXMJ19-His₆-prx) were harvested before and after H₂O₂ treatment for 10 min and subjected to the Ni-NTA His·Bind resin to enrich the His₆-CgPrx protein. The resulting His₆-CgPrx protein was demycothiolated using the CgMrx1/MSH/CgMtr-reducing system and the free protein thiol was tagged with biotin-maleimide, followed by separation on 15% nonreducing SDS-PAGE, and then analyzed with Western blotting by using the horseradish peroxidase-conjugated streptavidin to detect S-mycothiolation of CgPrx and the anti-His antibody to indicate the same amount of His₆-CgPrx used for demycothiolation analysis. (F) CgPrx had distinct stress-specific roles in resistance to heat stress. Survival rates of C. glutamicum wild type and Δprx mutant pretreated for 10 min with different concentrations of H₂O₂ to heat stress (45°C for 1 h) were determined. Note that the wild type pretreated with 150 mM, but not 50 mM, of H₂O₂ exhibited significantly increased resistance to heat stress. Mean values with standard deviations (error bars) from at least three repeats are shown. *p ≤ 0.05. IAM, iodoacetamide; Mrx1, mycoredoxin 1; MSH, mycothiol; Mtr, mycothiol disulfide reductase.

Treatment with 150 mM H₂O₂ resulted in the formation of sulfonated CgPrx tetramers as well as mycothiolated dimers. However, increasing H₂O₂ to 200 mM caused more production of sulfonated CgPrx tetramers (Fig. 4D). Withdrawing H₂O₂ for 120 min led to reduction of mycothiolated CgPrx (CgPrx-SSM) dimers, but not sulfonated CgPrx tetramers, indicating that CgPrx tetramers were irreversibly overoxidized to form sulfonic acid (Fig. 4A). Pretreatment of wild-type C. glutamicum with 150 mM H₂O₂ endowed significant resistance to subsequent heat stress treatment (45°C for 1h); such resistance did not occur in the Δprx mutant (Fig. 4F). In contrast, treatment with 50 mM H₂O₂ had no detectable effect in improving the heat stress resistance to either strain (Fig. 4F). Thus, sulfonated CgPrx tetramers induced by high levels of H₂O₂ possess chaperone activity.

Similar structure changes were observed for endogenous CgPrx treated under the same conditions visualized by a specific anti-CgPrx rabbit polyclonal antibody (Supplementary Fig. S5). These indicate that CgPrx confers different activities in different structural forms when the bacterial cells meet different levels of oxidative challenges.

Mycothiolation of Cys63 regulates the oligomerization and catalytic activity of CgPrx under moderate H₂O₂ stress

To further investigate the function of mycothiolated CgPrx under oxidative stress, we first analyzed the oligomeric status of His₆-CgPrx expressed in Δprx(pXMJ19-His₆-prx) and ΔprxΔmshC(pXMJ19-His₆-prx) under 120 mM H₂O₂ treatment. His₆-CgPrx from strain Δprx(pXMJ19-His₆-prx) exposed to 120 mM H₂O₂ showed a major band at about 40 kDa position (dimers), whereas His₆-CgPrx from similarly treated ΔprxΔmshC(pXMJ19-His₆-prx) hardly contained that 40 kDa band; instead, most of them existed as tetramers (Fig. 5A, lane 4). Consistently, dimer formation was also detected by treating purified CgPrx with 20 mM H₂O₂ and 0.5 mM MSH, but not with H₂O₂ alone (Fig. 5B). Instead, a band about 80 kDa corresponding to CgPrx tetramers was detected (Fig. 5B). A novel peak of 2605.9 Da that was 484 Da higher than that of the Cys-63-containing 56–99 peptide (2119.5 Da) in reduced CgPrx was observed in dimeric CgPrx (labeled [D] in Fig. 5A), indicating the addition of MSH on the Cys63 residue (Fig. 5D and Supplementary Fig. S4D).

FIG. 5.

S -mycothiolation reversibly protects CgPrx from overoxidation. (A) Proteins extracted from the Δprx(pXMJ19-His₆-prx) and ΔprxΔmshC(pXMJ19-His₆-prx) cells pretreated with 120 mM H₂O₂ were resolved on nonreducing SDS-PAGE and analyzed with Western blotting by using the anti-His antibody. (B) Purified His₆-CgPrx protein was pretreated with 20 mM H₂O₂ and 0.5 mM MSH or 20 mM H₂O₂ alone for 30 min, resolved on nonreducing SDS-PAGE, and then analyzed with Western blotting by using the anti-His antibody. (C) CgPrx-SSM and CgPrx were treated with 20 mM H₂O₂ for 30 min and resolved on nonreducing SDS-PAGE. (D, E) MALDI-TOF-TOF MS/MS analysis of dimeric and tetrameric CgPrx excised from nonreducing gels in (A) (labeled [D] and [E]). (F) Survival rates of recovered and not recovered cells (pretreated with 120 mM H₂O₂) to H₂O₂ (60 and 100 mM). During recovery, pretreated cells were incubated for 120 min in MM containing 25 μg/ml streptomycin. Mean values with standard deviations (error bars) from at least three repeats are shown. **p ≤ 0.01. (G) Peroxidase activity of CgPrx-SSM was determined in the presence of the CgTrx1-reducing system. The composition of the reaction system and the measuring method were the same as in Figure 1(A). (H) S-mycothiolated CgPrx could be reactivated by the CgMrx1 electron transfer pathway in vitro. The CgMrx1 electron transfer pathway comprising CgMrx1, MSH, CgMtr, and NADPH was reconstructed and CgPrx-SSM was added as the substrate. (I) Peroxidase activity of CgPrx-SSM was determined by measuring peroxide reduction with the Fox assay in the CgTrx/CgTrxR and CgMrx1/MSH/CgMtr-coupled enzyme assay system. Reaction mixtures contained 50 mM Tris-HCl buffer (pH 8.0), 2 mM ethylenediaminetetraacetic acid, 250 μM NADPH, 1 μM CgPrx-SSM, 5 μM CgTrxR, 40 μM CgTrx1, 5 μM CgMtr, 500 μM MSH, and 40 μM CgMrx1 in a final volume of 500 μl. The consumption of H₂O₂ was measured at indicated time points after H₂O₂ addition (100 μM). Reaction mixtures lacking either the CgTrx1 pathway, or the CgMrx1 pathway, or CgPrx-SSM in the assay system served as negative controls. Similar results were obtained in three independent experiments, and data shown are from one representative experiment done in triplicate. MM, mineral salts medium; Prx-SSM, mycothiolated Prx.

In contrast, mycothiolation of Cys63 was not observed in CgPrx tetramers. Instead, the Cys63 residue was sulfonated in CgPrx tetramers, with a mass of 2167.5 Da (labeled [E] in Fig. 5A) that was 48 Da higher than Cys63-containing 56–99 peptide of the reduced CgPrx (Fig. 5E). The above results suggest that mycothiolation of CgPrx prevents the formation of overoxidized tetramers under moderate oxidative stress. Consistent with this conclusion, while 20 mM H₂O₂-treated CgPrx appeared as both dimers and tetramers, similarly treated CgPrx-SSM existed only as dimers (Fig. 5C).

Protein S-thiolation is a reversible post-translational thiol modification that protects active site Cys residues of key enzymes against irreversible overoxidation and restores their functions under normal conditions (9). To investigate the role of S-mycothiolation in protecting the peroxidase activity of CgPrx, we determined the effects of recovery on the survival rates of low concentrations (60 and 100 mM) of H₂O₂-treated strains, Δprx(pXMJ19-prx) and ΔprxΔmshC(pXMJ19-prx). A 120-min recovery allowed the MSH-containing Δprx(pXMJ19-prx) strain pretreated with H₂O₂ to survive better. On the contrary, similar recovery time had very marginal effects on restoring the sensitivity phenotype of the MSH lacking ΔmshCΔprx(pXMJ19-prx) treated with H₂O₂ (Fig. 5F). Moreover, withdrawing H₂O₂ for 120 min led to a decrease of mycothiolated CgPrx dimers formed in the bacterial cells treated with 150 mM H₂O₂, which was accompanied by an increase in CgPrx monomers (Fig. 4A). These results indicate that S-mycothiolation not only regulates the oligomerization status of CgPrx but also reversibly protects its peroxidase activity under moderate H₂O₂ stress.

Mrx1-catalyzed demycothiolation reactivates S-mycothiolated CgPrx

S-mycothiolation inhibits the peroxidase activity of CgPrx in a way that can be restored by Mrx1-catalyzed demycothiolation. In agreement with this notion, no peroxidase activity was detected in S-mycothiolated CgPrx (CgPrx-SSM) (Fig. 5G). To reactivate mycothiolated CgPrx, we reconstituted the CgMrx1/CgMtr/MSH electron transfer pathway with CgPrx-SSM as a substrate. Only samples containing 50 μM CgPrx-SSM showed consumption of NADPH (Fig. 5H). No reduction of CgPrx-SSM was observed in the absence of CgMrx1, indicating that high levels of MSH (500 μM) alone were not sufficient to demycothiolate CgPrx-SSM in the absence of CgMrx1. As expected, demycothiolated CgPrx was able to restore the peroxidase activity in the CgTrx/CgTrxR and CgMrx1/CgMtr/MSH-coupled enzyme assay system (Fig. 5I). These results indicate that S-mycothiolation of Cys63 inhibits the peroxidase, and such inhibition can be reversed by demycothiolation with the CgMrx1/CgMtr/MSH-reducing system.

Consistent with this conclusion, a 120-min recovery significantly increased the survival rates of H₂O₂-pretreated Δprx(pXMJ19-prx) to low concentrations of H₂O₂ (60 and 100 mM) and had very marginal effects on restoring the sensitivity phenotype of H₂O₂-pretreated ΔprxΔmrx1(pXMJ19-prx) to the same treatment (Fig. 5F). In conclusion, S-mycothiolation reversibly protects CgPrx from overoxidation under moderate oxidative stress.

CgPrx modulates the activation of hydrogen peroxide-inducible gene activator and the expression of katA and mpx

In addition to Prx, KatA and MPx are two other major H₂O₂ scavengers in C. glutamicum. To distinguish the potentially different roles of Prx, KatA, and MPx in removing H₂O₂ by C. glutamicum, we investigated the survival rate of Δprx, Δmpx, and ΔkatA challenged with different H₂O₂ concentrations. Deletion of prx, mpx, or katA did not affect bacterial growth under normal condition without H₂O₂ stress (Supplementary Fig. S6A). Notably, the Δprx mutant was slightly, but substantially, more sensitive to low concentrations of H₂O₂ (45 mM) than the wild type, whereas the ΔkatA and Δmpx mutants showed wild-type-level resistance under the same condition. Conversely, the ΔkatA and Δmpx mutants were hypersensitive to high concentrations of H₂O₂ (100–200 mM) compared with the wild type, whereas the Δprx mutant showed a wild-type-level resistance under the same conditions (Fig. 6A). Consistent with the survival results, the ΔmpxΔkatA double mutant containing only Prx scavenged 5 mM H₂O₂ almost as effectively as the wild type; the ΔmpxΔprxΔkatA triple mutant completely failed to scavenge 120 mM H₂O₂. Whereas strains, ΔprxΔkatA and ΔprxΔmpx, were less effective as ΔmpxΔkatA in demolishing 5 mM H₂O₂, both were clearly more effective in abolishing 120 mM H₂O₂ than ΔmpxΔkatA (Fig. 6B). These results indicate that CgPrx, KatA, and MPx play distinct roles: CgPrx is more effective in scavenging very low concentrations of H₂O₂, while KatA and MPx are the more effective enzymes at higher concentrations. This conclusion was supported by the fact that the survival rates of the ΔmpxΔkatA mutant were significantly lower compared with the wild type after exposure to 120 mM H₂O₂ for 10 min; this mutant survived at rates similar to that of the wild type after exposure to 5 mM H₂O₂ for 30 min. In contrast, the ΔprxΔkatA and ΔprxΔmpx mutants lacking Prx survived at rates similar to that of the wild type after exposure to 120 mM H₂O₂ for 10 min; such rates were substantially lower compared with the wild type after exposure to 5 mM H₂O₂ for 30 min (Fig. 6C). Note that the growth of ΔprxΔkatA, ΔprxΔmpx, and ΔkatAΔmpx was nearly identical to the wild type under normal condition without H₂O₂ stress (Supplementary Fig. S6B).

FIG. 6.

Distinct roles of CgPrx, CgKatA, and CgMPx in scavenging H₂O₂. (A) Survival rates for WT, Δprx, Δmpx, and ΔkatA strains after being challenged with various concentrations of H₂O₂. Mean values with standard deviations (error bars) from at least three repeats are shown. *p ≤ 0.05 and **p ≤ 0.01. (B) Distinct efficiencies of CgPrx, CgMPx, and CgKatA in scavenging H₂O₂. H₂O₂ was added at a final concentration of 5 mM (left panel) and 120 mM (right panel) to cultures of WT, ΔmpxΔkatA, ΔmpxΔprx, ΔprxΔkatA, and ΔmpxΔprxΔkatA strains grown to A ₆₀₀ = 1.6–1.7 in MM containing glucose. At various time points after addition of H₂O₂, the H₂O₂ concentration was measured with the FOX assay. (C) Survival rates of ΔmpxΔkatA, ΔprxΔmpx, ΔprxΔkatA, and ΔmpxΔprxΔkatA strains after being challenged with 5 mM H₂O₂ for 30 min or 120 mM H₂O₂ for 10 min. Mean values with standard deviations (error bars) from at least three repeats are shown. **p ≤ 0.01; *p ≤ 0.05. (D) Effects of prx deletion on the promoter activities of katA (upper panel) and mpx (lower panel) under various concentrations of H₂O₂. Indicated reporter strains were grown in MM to exponential phase, and β-galactosidase activities were measured 10 min after exposure to indicated concentrations of H₂O₂. (E) Effects of prx deletion on OxyR activation under various concentrations of H₂O₂. His₆-OxyR was extracted from the WT(pXMJ19-His₆-oxyR) and Δprx(pXMJ19-His₆-oxyR) cells treated with indicated H₂O₂ concentrations for 10 min, modified with AMS, resolved on nonreducing SDS-PAGE, and then detected by Western blotting with the anti-His antibody. FOX, ferrous xylenol orange; KatA, catalase; MPx, mycothiol peroxidase; OxyR, hydrogen peroxide-inducible gene activator. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Consistent with the above conclusions, the expression level of katA and mpx was lower than that of prx under low concentrations of H₂O₂, but became significantly higher than that of prx in the presence of high concentrations of H₂O₂ (Fig. 6D). Strikingly, the expression level of katA and mpx increased to that of prx level at low concentrations of H₂O₂ in a strain without the prx gene (Fig. 6D), suggesting that Prx inhibits the expression of katA and mpx under low H₂O₂ concentration, possibly by keeping an extremely low intracellular H₂O₂ concentration through its peroxidase activity. An increase in intracellular H₂O₂ levels is known to signal the activation of the primary redox-sensitive regulator hydrogen peroxide-inducible gene activator (OxyR) by oxidation, leading to the induction of the expression of the more powerful KatA (41) and also possibly MPx. Indeed, at least 50 mM H₂O₂ was required for OxyR activation in wild-type bacteria, but only 25 mM was required for such activation in the Δprx mutant, which was sufficient to induce the expression of katA and possibly mpx (Fig. 6E).

Discussion

The results herein revealed that CgPrx, an atypical bacterial 2-Cys Prx, by acting as a delicate H₂O₂ sensor and the primary scavenger of H₂O₂ in C. glutamicum, indeed plays central roles for making appropriate responses to different levels of H₂O₂ (Fig. 7). (i) Under low peroxide concentrations, the H₂O₂-sensing peroxidatic Cys63 forms an intramolecular disulfide with the resolving Cys97, which allows monomeric CgPrx to reduce peroxide as a Trx-dependent peroxidase. This activity prevents KatA and MPx from unnecessary expression. (ii) Moderate peroxide challenge induces the reversible S-mycothiolation of the H₂O₂-sensing Cys63, converting CgPrx into an inactive dimer form. Thus, increased levels of H₂O₂ oxidize redox-sensitive regulators such as OxyR to induce the expression of the more efficient KatA and MPx to eliminate the excess peroxide. (iii) Under hyperoxidative stress condition, the H₂O₂-sensing Cys63 will be hyperoxidized to Cys-sulfonate, leading to irreversible inactivation of peroxidase activity. Most CgPrx molecules will transit to tetrameric and higher molecular weight chaperones for the salvage of damaged proteins.

FIG. 7.

Proposed reaction mechanisms for CgPrx in C. glutamicum . CgPrx plays central roles for making appropriate responses to different levels of H₂O₂. At low H₂O₂ concentration, the peroxidatic Cys (Cys63, C_P) catalyzes the reduction of H₂O₂ to H₂O, resulting in the formation of an intramolecular disulfide with the resolving cysteine (Cys97, C_R). The monomeric CgPrx disulfide is reduced by thioredoxin to regenerate reduced CgPrx. This activity prevents catalase and MPx from unnecessary expression. Moderate peroxide challenge induces the reversible S-mycothiolation of the H₂O₂-sensing Cys63, thus converting CgPrx into an inactive dimer form and allowing increased levels of intracellular H₂O₂ to activate redox-sensitive regulators such as OxyR, then inducing the expression of the more efficient catalase and MPx to eliminate excess peroxide. Under hyperoxidative stress condition, the H₂O₂-sensing Cys63 will be hyperoxidized to Cys-sulfonate, leading to irreversible inactivation of peroxidase activity. Most CgPrx molecules will transit to tetrameric and higher molecular weight chaperones for salvage of damaged proteins. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

The typical 2-Cys Prx is able to assume multiple oligomeric structures in response to cellular redox states and the transition between these structures dynamically regulates the switch between the peroxidase and chaperone activities (5). For example, the 2-Cys Prx from yeast transits from LMW species to oligomeric and HMW complexes upon being challenged by oxidative stress or heat shock, whose activity is switched from peroxidase to chaperone (18). Although atypical 2-Cys Prxs appear to exist in the lower polymerization degree (dimers, tetramers, and hexamers) compared with typical 2-Cys Prxs (decamers and HMW species), increasing evidence indicates that they also undergo dynamic conformation transitions with functional implications (4, 5). Our finding that high concentrations of H₂O₂ induce CgPrx into dimers, tetramers, and even HMW species is in line with these observations. While most of typical 2-Cys Prxs and even some 1-Cys Prxs have been found to function as both peroxidases and molecular chaperones (1, 2, 8, 18, 22), this is the first report on the dual activity of an atypical 2-Cys Prx.

Besides being hyperoxidized to induce the peoxidase-to-chaperone functional switch under high H₂O₂ concentrations, the Cys63 sensor was modified by S-mycothiolation under moderate concentrations of H₂O₂ treatment (Fig. 4). S-mycothiolation clearly protects CgPrx from being damaged under oxidative stress, which is in line with the fact that a reversible thiol modification often protects active Cys of key enzymes against irreversible overoxidation to sulfonic acids. In addition, S-mycothiolation plays a crucial role in regulating the oligomerization status of CgPrx in a way different from S-glutathionylation. While S-glutathionylation induced the dissociation of Prx oligomers (6, 32, 36), S-mycothiolation maintains CgPrx exclusively in its dimeric form, defective in both peroxidase and chaperone activity, and prevents the CgPrx dimer to form tetramer and HMW complexes (Fig. 5). Thus, S-mycothiolation of CgPrx not only maintains CgPrx in its dimeric form, defective in both peroxidase and chaperone activity, but also protects CgPrx from irreversible overoxidation.

In eukaryotes, 2-Cys Prxs containing the oxidative susceptible GGLG and YF motifs utilize a unique regulatory mechanism to modulate the peroxide-mediated signaling cascades. According to the floodgate hypothesis, sensitive 2-Cys Prxs act as a peroxide floodgate to keep peroxides away from susceptible molecules until it is opened by inactivation via overoxidation, which allows a rapid increase in H₂O₂ levels and thus permits its signaling activity (51). In contrast, because of the lack of the GGLG and YF motifs and their robust response to oxidative inactivation, bacterial 2-Cys Prxs have never been attributed to signaling events. Strikingly, our results clearly demonstrate that CgPrx uses a more complex floodgate mechanism to modulate the peroxide signal in C. glutamicum. Inactivation of CgPrx by S-mycothiolation induced the expression of katA and mpx via activating the OxyR regulator. This mechanism provides a rapid and reversible way to open the floodgate without the need to produce the irreversibly inactivated protein. The CgPrx floodgate can also be completely opened by irreversible overoxidation, which occurs possibly under severe oxidative conditions that threaten the viability of bacterial cells. Thus, it appears that bacterial Prx has evolved as a more complex mechanism than ever thought before for adapting appropriate responses to different levels of oxidative challenge.

Materials and Methods

Bacterial strains and culture conditions

Bacterial strains and plasmids used in this study are listed in Supplementary Table S1. C. glutamicum and Escherichia coli strains were cultured in mineral salts medium (MM) or Luria-Bertani (LB) medium as previously reported (43). In-frame deletions were generated by means of the method described (45). Sensitivity assays for peroxides were performed as described (45).

Cloning, expression, and purification of recombinant proteins

The genes encoding C. glutamicum Trx2 (NCgl2874), Trx3 (NCgl0289), CgPrx (NCgl1041), and OxyR (NCgl1850), were amplified by polymerase chain reaction (PCR). These DNA fragments were digested and subcloned into similar digested vectors, obtaining pET28a-trx2, pET28a-trx3, pET28a-prx, pGEX6p-1-prx, pXMJ19-prx, pXMJ19-His₆-prx, and pXMJ19-His₆-oxyR, respectively. Site-directed mutagenesis was constructed as described (53). The plasmids, pK18mobsacB-Δprx and pK18mobsacB-ΔkatA, used to construct deletion mutants were made by overlap PCR (53). The lacZ fusion reporter vectors, pK18mobsacB-P_katA::lacZ and pK18mobsacB-P_prx::lacZ, were made by fusion of the katA and prx promoter to the lacZY reporter gene via overlap PCR (53). For overexpression or complementation in C. glutamicum strains, corresponding pXMJ19 derivatives were transformed into C. glutamicum by electroporation. The transformants were selected on LB plates supplemented with nalidixic acid and chloramphenicol (45). Expression in C. glutamicum was induced by addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside. To express and purify GST- and His₆-tagged recombinant proteins in E. coli, the pGEX6p-1 and pET28a derivatives were transformed into E. coli XL1Blue and BL21(DE3), respectively. Recombinant proteins were purified as described (52). Primers used in this study are listed in Supplementary Table S2. The fidelity of all constructs was confirmed by DNA sequencing.

Analysis of sulfenic acid and disulfide bond formation

The formation of Cys-SOH as a reaction intermediate was examined by the assays of 50 mM DTT-treated or 10 mM H₂O₂-treated CgPrx:C84SC97S, CgPrx:C63SC84S, and CgPrx:C63SC97S proteins labeled with NBD-Cl (12, 42). The formation of disulfide bond was measured with AMS (Molecular Probes, Eugene, OR) as described (11).

Enzyme assays

Peroxidase activity of CgPrx was determined by monitoring the rate of NADPH oxidation (3). By using the Trx-based regeneration system, the reaction mixture comprised 50 mM Tris-HCl buffer (pH 8.0), 2 mM ethylenediaminetetraacetic acid (EDTA), 250 μM NADPH, 5 μM CgTrxR, 1 μM CgPrx, and 40 μM CgTrx. In alternative assays by using the CgMrx1/CgMtr/MSH-based system, CgTrxR was replaced by 5 μM CgMtr and 500 μM MSH and CgTrx was replaced by 40 μM CgMrx1. The reaction was started by addition of 100 μM peroxide, following a 5-min preincubation, and NADPH oxidation was monitored at 340 nm. Molecular chaperone activities were determined by assessing the ability of recombinant CgPrx to inhibit the aggregation of MDH and insulin substrate as described previously (1, 2). β-Galactosidase assay was performed with o-nitrophenyl β-d-galactopyranoside as the substrate (26).

Measurement of intracellular ROS levels and determination of cellular protein carbonylation

Intracellular ROS levels were measured using H₂DCFDA (48). Protein carbonylation assays were performed as described (47).

Oxidative stress-dependent structural switch of CgPrx in vivo

To determine oxidative stress-dependent structural switch of CgPrx in vivo, C. glutamicum wild-type, Δprx(pXMJ19-His₆-prx), and ΔmshCΔprx(pXMJ19-His₆-prx) strains were cultured in MM containing 1% glucose at 30°C. Bacterial cells were grown to mid-exponential phase and split into 80-ml aliquots for H₂O₂ treatment (50–200 mM, 10 min). The stressed samples were harvested immediately by centrifugation. For recovery, the stressed samples were inoculated into fresh MM containing 25 μg/ml streptomycin at 30°C for 2 h and then harvested. Crude cell lysates were subjected to nonreducing SDS-PAGE, and the structural properties of CgPrx were visualized by immunoblotting using anti-His antibody and analyzed by MALDI-TOF-TOF MS/MS (Voyager-DE STR; Applied Biosystems, Waltham, MA). Structure properties were also visualized for endogenous CgPrx by using an anti-CgPrx rabbit polyclonal antibody generated and affinity purified according to the method described previously (16).

Mycothiolation of CgPrx in vivo and in vitro

Iodoacetamide (IAM)-biotin-tagged demycothiolated proteins were obtained as described (17). Briefly, Δprx(pXMJ19-His₆-prx) and ΔmshCΔprx(pXMJ19-His₆-prx) strains were grown aerobically to an A ₆₀₀ of 0.9. Each culture was divided into two parts, one was exposed to 120 mM H₂O₂ for 10 min at 30°C, while the other was grown without H₂O₂ (negative control). Cells were harvested and resuspended in urea/3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate (CHAPS) alkylation buffer (100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 8 M urea, 1% CHAPS, 100 mM IAM) for 30 min in the dark before sonication on ice. Protein extracts were obtained by centrifugation and continued to be alkylated for 30 min in the dark. His₆-Prx was purified using His·Bind Ni-NTA resin (Novagen, Madison, WI) in accordance with the manufacturer's instructions. Purified His₆-Prx was demycothiolated using 25 μM recombinant Mrx1 in the presence of 1 mM MSH, 250 μM NADPH, and 5 μM Mtr for 30 min at room temperature. The mixture was then treated with 5 mM biotin-maleimide dissolved in dimethyl sulfoxide for 30 min. Unreacted biotin-maleimide was removed by ice-cold acetone precipitation three times for 1 h, followed by centrifugation at 10,000 g for 30 min. The pellet was redissolved in Tris-HCl buffer, separated using nonreducing SDS-PAGE, and detected by Western blotting with streptavidin-horseradish peroxidase (1:300; Thermo, Rockford, IL). The resulting His₆-Prx (10 μM) was also separated by reducing SDS-PAGE and detected by Western blotting with anti-His antibody or stained with Coomassie Brilliant Blue. Protein bands were excised from the gel, in-gel digested with trypsin, and analyzed using MALDI-TOF-TOF MS/MS. In vitro mycothiolation of CgPrx was conducted according to the method of Chi et al. (7). MSH was purified from C. glutamicum as described (13).

H₂O₂ scavenging by whole cells

C. glutamicum strains were grown to stationary phase in MM containing 1% glucose at 30°C. Then, 5 and 120 mM H₂O₂ was added, respectively, and continued to incubate at 30°C in 100 rpm. At intervals, aliquots were immediately harvested, diluted when necessary, and assayed for H₂O₂ content by the ferrous xylenol orange assay (49).

Redox response of OxyR

WT(pXMJ19-His₆-oxyR) and Δprx(pXMJ19-His₆-oxyR) strains were grown to stationary phase and treated with different concentrations of H₂O₂ for 10 min at 30°C. Proteins extracted from the cells with TissueLyser II (Qiagen, Hilden, Germany) were reacted with 15 mM AMS in the dark (11), resolved on nonreducing SDS-PAGE, and then detected by Western blotting with anti-His antibody.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31270078 and 31500087), Key Science and Technology R&D Program of Shaanxi Province, China (2014K02-12-01), and the Natural Science Foundation of Shandong Province, China (ZR2015CM012).

Author Disclosure Statement

No competing financial interests exist.

Abbreviations Used

References

, Lee

, Wi

, Jung

, Park

, and Chung

. New antioxidant with dual functions as a peroxidase and chaperone in Pseudomonas aeruginosa . Mol Cells, 29: 145–151, 2010.

, Lee

, Wi

, Jung

, Park

, Lee

, and Chung

. Functional switching of a novel prokaryotic 2-Cys peroxiredoxin (PpPrx) under oxidative stress. Cell Stress Chaperones, 16: 317–328, 2011.

Baker

LMS

and Poole

. Catalytic mechanism of thiol peroxidase from Escherichia coli—sulfenic acid formation and overoxidation of essential CYS61. J Biol Chem, 278: 9203–9211, 2003.

Barranco-Medina

, Kakorin

, Lazaro

, and Dietz

. Thermodynamics of the dimer-decamer transition of reduced human and plant 2-cys peroxiredoxin. Biochemistry, 47: 7196–7204, 2008.

Barranco-Medina

, Lázaro

, and Dietz

. The oligomeric conformation of peroxiredoxins links redox state to function. FEBS Lett, 583: 1809–1816, 2009.

Chae

, Oubrahim

, Park

, Rhee

, and Chock

. Protein glutathionylation in the regulation of peroxiredoxins: a family of thiol-specific peroxidases that function as antioxidants, molecular chaperones, and signal modulators. Antioxid Redox Signal, 16: 506–523, 2012.

Chi

, Busche

, Van Laer

, Basell

, Becher

, Clermont

, Seibold

, Persicke

, Kalinowski

, Messens

, and Antelmann

. Protein S-mycothiolation functions as redox-switch and thiol protection mechanism in Corynebacterium glutamicum under hypochlorite stress. Antioxid Redox Signal, 20: 589–605, 2013.

Chuang

, Wu

, Lo

, Lin

, Wong

, and Chiou

. The antioxidant protein alkylhydroperoxide reductase of Heliclobacter pylori switches from a peroxide reductase to a molecular chaperone function. Proc Natl Acad Sci U S A, 103: 2552–2557, 2006.

Dalle-Donne

, Milzani

, Gagliano

, Colombo

, Giustarini

, and Rossi

. Molecular mechanisms and potential clinical significance of S-glutathionylation. Antioxid Redox Signal, 10: 445–473, 2008.

10.

Day

, Brown

, Taylor

, Rand

, Morgan

, and Veal

. Inactivation of a peroxiredoxin by hydrogen peroxide is critical for thioredoxin-mediated repair of oxidized proteins and cell survival. Mol Cell, 45: 398–408, 2012.

11.

Ehira

and Ohmori

. The redox-sensing transcriptional regulator RexT controls expression of thioredoxin A2 in the Cyanobacterium anabaena sp. strain PCC 7120. J Biol Chem, 287: 40433–40440, 2012.

12.

Ellis

and Poole

. Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. Biochemistry, 36: 15013–15018, 1997.

13.

Feng

, Che

, Milse

, Yin

, Liu

, Ruckert

, Shen

, Qi

, Kalinowski

, and Liu

. The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum . J Biol Chem, 281: 10778–10785, 2006.

14.

Fujii

and Ikeda

. Advances in our understanding of peroxiredoxin, a multifunctional, mammalian redox protein. Redox Rep, 3: 123–129, 2002.

15.

Hall

, Karplus

, and Poole

. Typical 2-Cys peroxiredoxins-structures, mechanisms and functions. FEBS J, 276: 2469–2477, 2009.

16.

Hubber

, Arasaki

, Nakatsu

, Hardiman

, Lambright

, de Camilli

, Nagai

, and Roy

. The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins. PLoS Pathog, 10: e1004222, 2014.

17.

Jaffrey

and Snyder

. The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE, 86: pl1, 2001.

18.

Jang

, Lee

, Chi

, Jung

, Park

, Lee

, Moon

, Yun

, Choi

, Kim

, Kang

, Cheong

, Yun

, Rhee

, Cho

, and Lee

. Two enzymes in one: two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function. Cell, 117: 625–635, 2004.

19.

Karplus

. A primer on peroxiredoxin biochemistry. Free Radic Biol Med, 80: 183–190, 2015.

20.

Knoops

, Goemaere

, Van der Eecken

, and Declercq

. Peroxiredoxin 5: structure, mechanism, and function of the mammalian atypical 2-Cys peroxiredoxin. Antioxid Redox Signal, 15: 817–829, 2011.

21.

Lee

, Iijima-Ando

, Iijima

, Lee

, Yu

, and Lee

. JNK/FOXO-mediated neuronal expression of fly homologue of peroxiredoxin II reduces oxidative stress and extends life span. J Biol Chem, 284: 29454–29461, 2009.

22.

Lee

, Jia

, Liu

, Pham

, Kwak

, Xuan

, and Cheong

. A 1-Cys peroxiredoxin from a thermophilic archaeon moonlights as a molecular chaperone to protect protein and DNA against stress-induced damage. PLoS One, 10: e0125325, 2015.

23.

Liu

, Long

, Yin

, Si

, Zhang

, Lu

, Wang

, and Shen

. Physiological roles of mycothiol in detoxification and tolerance to multiple poisonous chemicals in Corynebacterium glutamicum . Arch Microbiol, 195: 419–429, 2013.

24.

Matte

, De Falco

, Iolascon

, Mohandas

, An

, Siciliano

, Leboeuf

, Janin

, Bruno

, Choi

, Kim

, and De Franceschi

. The interplay between peroxiredoxin-2 and nuclear factor-erythroid 2 is important in limiting oxidative mediated dysfunction in β-thalassemic erythropoiesis. Antioxid Redox Signal, 23: 1284–1297, 2015.

25.

Mieyal

and Chock

. Posttranslational modification of cysteine in redox signaling and oxidative stress: focus on S-glutathionylation. Antioxid Redox Signal, 16: 471–475, 2012.

26.

Miller

. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Plainview, NY: Cold Spring Harbor Laboratory Press, 1992.

27.

Milse

, Petri

, Ruckert

, and Kalinowski

. Transcriptional response of Corynebacterium glutamicum ATCC 13032 to hydrogen peroxide stress and characterization of the OxyR regulon. J Biotechnol, 190: 40–54, 2014.

28.

Monteiro

, Horta

, Pimenta

, Augusto

, and Netto

. Reduction of 1-Cys peroxiredoxins by ascorbate changes the thiol-specific antioxidant paradigm, revealing another function of vitamin C. Proc Natl Acad Sci U S A, 104: 4886–4891, 2007.

29.

Moon

, Hah

, Kim

, Jung

, Jang

, Lee

, Kim

, Lee

, Jeon

, Kim

, Cho

, and Lee

. Oxidative stress-dependent structural and functional switching of a human 2-Cys peroxiredoxin isotype II that enhances HeLa cell resistance to H₂O₂-induced cell death. J Biol Chem, 280: 28775–28784, 2005.

30.

Morais

MAB

, Giuseppe

, Souza

TACB

, Alegria

TGP

, Oliveira

, Netto

LES

, and Murakami

. How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily. J Biol Chem, 290: 8582–8590, 2015.

31.

Newton

, Buchmeier

, and Fahey

. Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria . Microbiol Mol Biol Rev, 72: 471–494, 2008.

32.

Noguera-Mazon

, Lemoine

, Walker

, Rouhier

, Salvador

, Jacquot

, Lancelin

, and Krimm

. Glutathionylation induces the dissociation of 1-Cys D-peroxiredoxin non-covalent homodimer. J Biol Chem, 281: 31736–31742, 2006.

33.

Nystrom

, Yang

, and Molin

. Peroxiredoxins, gerontogenes linking aging to genome instability and cancer. Genes Dev, 26: 2001–2008, 2012.

34.

Olahova

, Taylor

, Khazaipoul

, Wang

, Morgan

, Matsumoto

, Blackwell

, and Veal

. A redox-sensitive peroxiredoxin that is important for longevity has tissue- and stress-specific roles in stress resistance. Proc Natl Acad Sci U S A, 105: 19839–19844, 2008.

35.

Ordóñez

, Van Belle

, Roos

, De Galan

, Letek

, Gil

, Wyns

, Mateos

, and Messens

. Arsenate reductase, mycothiol, and mycoredoxin concert thiol/disulfide exchange. J Biol Chem, 284: 15107–15116, 2009.

36.

Park

, Piszczek

, Rhee

, and Chock

. Glutathionylation of peroxiredoxin I induces decamer to dimers dissociation with concomitant loss of chaperone activity. Biochemistry, 50: 3204–3210, 2011.

37.

Pedrajas

, McDonagh

, Hernandez-Torres

, Miranda-Vizuete

, Gonzalez-Ojeda

, Martinez-Galisteo

, Padilla

, and Barcena

. Glutathione is the resolving thiol for thioredoxin peroxidase activity of 1-Cys peroxiredoxin without being consumed during the catalytic cycle. Antioxid Redox Signal, 24: 115–128, 2016.

38.

Pedre

, Van Molle

, Villadangos

, Wahni

, Vertommen

, Turell

, Erdogan

, Mateos

, and Messens

. The Corynebacterium glutamicum mycothiol peroxidase is a reactive oxygen species-scavenging enzyme that shows promiscuity in thiol redox control. Mol Microbiol, 96: 1176–1191, 2015.

39.

Perkins

, Poole

, and Karplus

. Tuning of peroxiredoxin catalysis for various physiological roles. Biochemistry, 53: 7693–7705, 2014.

40.

Rhee

and Woo

. Multiple functions of peroxiredoxins: peroxidases, sensors and regulators of the intracellular messenger H₂O₂, and protein chaperones. Antioxid Redox Signal, 15: 781–794, 2011.

41.

Seaver

and Imlay

. Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli . J Bacteriol, 183: 7173–7181, 2001.

42.

Selles

, Hugo

, Trujillo

, Srivastava

, Wingsle

, Jacquot

, Radi

, and Rouhier

. Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5: kinetics, catalytic mechanism and oxidative inactivation. Biochem J, 442: 369–380, 2012.

43.

Shen

, Jiang

, Huang

, Liu

, and Liu

. Functional identification of novel genes involved in the glutathione-independent gentisate pathway in Corynebacterium glutamicum . Appl Environ Microbiol, 71: 3442–3452, 2005.

44.

Shen

, Zhou

, and Liu

. Degradation and assimilation of aromatic compounds by Corynebacterium glutamicum: another potential for applications for this bacterium?. Appl Microbiol Biotechnol, 95: 77–89, 2012.

45.

, Xu

, Wang

, Long

, Ding

, Chen

, Guan

, Liu

, Wang

, Shen

, and Liu

. Functional characterization of a mycothiol peroxidase in Corynebacterium glutamicum that uses both mycoredoxin and thioredoxin reducing systems in the response to oxidative stress. Biochem J, 469: 45–57, 2015.

46.

Teramoto

, Inui

, and Yukawa

. OxyR acts as a transcriptional repressor of hydrogen peroxide-inducible antioxidant genes in Corynebacterium glutamicum . FEBS J, 280: 3298–3312, 2013.

47.

Vinckx

, Wei

, Matthijs

, Noben

, Daniels

, and Cornelis

. A proteome analysis of the response of a Pseudomonas aeruginosa OxyR mutant to iron limitation. Biometals, 24: 523–532. 2011

48.

Wang

, Si

, Song

, Zhu

, Gao

, Wang

, Zhang

, Wei

, Luo

, and Shen

. Type VI secretion system transports Zn²⁺ to combat multiple stresses and host immunity. PLoS Pathog, 11: e1005020, 2015.

49.

Wolff

. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Method Enzymol, 233: 182–189, 1994.

50.

Woo

, Yim

, Shin

, Kang

, Yu

, and Rhee

. Inactivation of peroxiredoxin I by phosphorylation allows localized H₂O₂ accumulation for cell signaling. Cell, 140: 517–528, 2010.

51.

Wood

, Poole

, and Karplus

. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science, 300: 650–653, 2003.

52.

, Shen

, Bryan

, Banga

, Swanson

, and Luo

. Inhibition of host vacuolar H⁺-ATPase activity by a Legionella pneumophila effector. PLoS Pathog, 6: e1000822. 2010.

53.

Zhang

, Wang

, Song

, Wang

, Xu

, Peng

, Lin

, Zhang

, and Shen

. A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis. Environ Microbiol, 15: 557–569, 2013.

Supplementary Material

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Graded Response of the Multifunctional 2-Cysteine Peroxiredoxin,CgPrx,to Increasing Levels of Hydrogen Peroxide in Corynebacterium glutamicum

Abstract

Aims:

Results:

Innovation:

Conclusion:

Introduction

Results

CgPrx is required for resistance to low oxidative stress

Cys63 and Cys97 form an intramolecular disulfide bond during the reduction of H2O2

CgPrx switches from a peroxidase to a molecular chaperone upon sulfonation of Cys63 by high concentrations of H2O2

Oxidative stress-induced structural changes of CgPrx in vivo

Mycothiolation of Cys63 regulates the oligomerization and catalytic activity of CgPrx under moderate H2O2 stress

Mrx1-catalyzed demycothiolation reactivates S-mycothiolated CgPrx

CgPrx modulates the activation of hydrogen peroxide-inducible gene activator and the expression of katA and mpx

Discussion

Materials and Methods

Bacterial strains and culture conditions

Cloning, expression, and purification of recombinant proteins

Analysis of sulfenic acid and disulfide bond formation

Enzyme assays

Measurement of intracellular ROS levels and determination of cellular protein carbonylation

Oxidative stress-dependent structural switch of CgPrx in vivo

Mycothiolation of CgPrx in vivo and in vitro

H2O2 scavenging by whole cells

Redox response of OxyR

Footnotes

Acknowledgments

Author Disclosure Statement

Abbreviations Used

References

Supplementary Material

Cys63 and Cys97 form an intramolecular disulfide bond during the reduction of H₂O₂

CgPrx switches from a peroxidase to a molecular chaperone upon sulfonation of Cys63 by high concentrations of H₂O₂

Mycothiolation of Cys63 regulates the oligomerization and catalytic activity of CgPrx under moderate H₂O₂ stress

H₂O₂ scavenging by whole cells