The Corynebacterium glutamicum mycothiol peroxidase is a reactive oxygen species‐scavenging enzyme that shows promiscuity in thiol redox control

Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (‐SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H₂O₂ reduction, a sulfenic acid (‐SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S‐S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx‐MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H₂O₂ stress, and its gene expression is clearly induced upon H₂O₂ challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.     

Corynebacterium diphtheriae Methionine Sulfoxide Reductase A Exploits a Unique Mycothiol Redox Relay Mechanism

Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.     

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H₂O₂-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.     

Mycobacterium tuberculosis Rv3034c regulates mTORC1 and PPAR‐γ dependant pexophagy mechanism to control redox levels in macrophages

Mycobacterium tuberculosis survives inside the macrophages by employing several host immune evasion strategies. Here, we reported a novel mechanism in which M. tuberculosis acetyltransferase, encoded by Rv3034c, induces peroxisome homeostasis to regulate host oxidative stress levels to facilitate intracellular mycobacterial infection. Presence of M. tuberculosis Rv3034c induces the expression of peroxisome biogenesis and proliferation factors such as Pex3, Pex5, Pex19, Pex11b, Fis‐1 and DLP‐1; while depletion of Rv3034c decreased the expression of these molecules, thereby selective degradation of peroxisomes via pexophagy. Further studies revealed that M. tuberculosis Rv3034c inhibit induction of pexophagy mechanism by down‐regulating the expression of pexophagy associated proteins (p‐AMPKα, p‐ULK‐1, Atg5, Atg7, Beclin‐1, LC3‐II, TFEB and Keap‐1) and adaptor molecules (NBR1 and p62). Inhibition was found to be dependent on the phosphorylation of mTORC1 and activation of peroxisome proliferator activated receptor‐γ. In order to maintain intracellular homeostasis during oxidative stress, M. tuberculosis Rv3034c was found to induce degradation of dysfunctional and damaged peroxisomes through activation of Pex14 in infected macrophages. In conclusion, this is the first report which demonstrated that M. tuberculosis acetyltransferase regulate peroxisome homeostasis in response to intracellular redox levels to favour mycobacterial infection in macrophage.     

The concerted action of a positive charge and hydrogen bonds dynamically regulates the pKa of the nucleophilic cysteine in the NrdH‐redoxin family

NrdH‐redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH‐redoxins have a CVQC active site motif and belong to the thioredoxin‐fold protein family. As for other thioredoxin‐fold proteins, the pK_a of the nucleophilic cysteine of NrdH‐redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the pK_a value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH‐redoxins were determined, but structural insights explaining the relatively low pK_a remained elusive. We subjected C. glutamicum NrdH‐redoxin to an extensive molecular dynamics simulation to expose the factors regulating the pK_a of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N‐terminal of the active site further lowers the cysteine pK_a. However, site‐directed mutagenesis data show that the major contribution to the lowering of the cysteine pK_a comes from the positive charge of the lysine and not from the additional Lys‐Cys hydrogen bond. In 12% of the NrdH‐redoxin family, this lysine is replaced by an arginine that also lowers the cysteine pK_a. All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N‐terminally of the active site dynamically regulate the pK_a of the nucleophilic cysteine in NrdH‐redoxins.     

Inositol monophosphate phosphatase genes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.     

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.     

Glutathione disulfide and S-nitrosoglutathione detoxification by Mycobacteriumtuberculosis thioredoxin system

Mycobacterium tuberculosis resides within alveolar macrophages. These phagocytes produce reactive nitrogen and oxygen intermediates to combat the invading pathogens. The macrophage glutathione (GSH) pool reduces nitric oxide (NO) to S-nitrosoglutathione (GSNO). Both glutathione disulfide (GSSG) and GSNO possess mycobactericidal activities in vitro. In this study we demonstrate that M. tuberculosis thioredoxin system, comprises of thioredoxin reductase B2 and thioredoxin C reduces the oxidized form of the intracellular mycothiol (MSSM) and is able to efficiently reduce GSSG and GSNO in vitro. Our study suggests that the thioredoxin system provide a general reduction mechanism to cope with oxidative stress associated with the microbe’s metabolism as well as to detoxify xenobiotics produced by the host.     

Mycothiol-dependent mycobacterial response to oxidative stress

The effect of exogenous oxidative stress on mycothiol (MSH) levels and redox balance was investigated in mycobacteria. Both the thiol-specific oxidant diamide and hydrogen peroxide induced up to 75% depletion of MSH to form the disulfide form, mycothione (MSSM), in Mycobacterium bovis BCG. In comparison, Mycobacterium smegmatis, a saprophytic mycobacterium, displays a greater tolerance towards these oxidants, reflected by the lack of fluxes in MSH levels and redox ratios upon oxidative stress treatments. The basal ratio of MSH to MSSM was established to be 50:1 in M. bovis BCG and 200:1 in M. smegmatis.     

Solution structures of Mycobacterium tuberculosis thioredoxin C and models of intact thioredoxin system suggest new approaches to inhibitor and drug design

Here, we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C‐terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared with the solution structure. On the basis of these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti‐mycobacterial agents, or as chemical genetic probes of function. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

A novel F420‐dependent anti‐oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents

Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F₄₂₀‐dependent anti‐oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F₄₂₀‐deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD⁺ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F₄₂₀^? mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin‐dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F₄₂₀H₂‐dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F₄₂₀H₂‐specific obligate two‐electron reduction of endogenous quinones, thereby competing with the one‐electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F₄₂₀ biosynthesis pathway or Fqr‐class proteins as a mechanism to potentiate the action of bactericidal agents.     

CysK2 from Mycobacterium tuberculosis Is an O-Phospho-l-Serine-Dependent S-Sulfocysteine Synthase

Mycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to l-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), have been described in M. tuberculosis, but the function of a third putative sulfhydrylase in this pathogen, CysK2, has remained elusive. We present biochemical and biophysical evidence that CysK2 is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. The enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as k_max/K_s = (3.97 × 10³) ± 619 M⁻¹ s⁻¹, which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a k_max/K_s of (1.34 × 10³) ± 48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant k_cat/K_m for thiosulfate is 40-fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine. Hypothetically, S-sulfocysteine could also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy.     

Structural and mechanistic insights into Mycothiol Disulphide Reductase and the Mycoredoxin-1-alkylhydroperoxide reductase E assembly of Mycobacterium tuberculosis

Mycobacteria employ a versatile machinery of the mycothiol-dependent system, containing the proteins mycothiol disulfide reductase (Mtr), the oxido-reductase Mycoredoxin-1 (Mrx-1) and the alkyl-hydroperoxide subunit E (AhpE). The mycothiol-dependent protein ensemble regulates the balance of oxidized-reduced mycothiol, to ensure a reductive intracellular environment for optimal functioning of its proteins even upon exposure to oxidative stress. Here, we determined the first low-resolution solution structure of Mycobacterium tuberculosis Mtr (MtMtr) derived from small-angle X-ray scattering data, which provides insight into its dimeric state. The solution shape reveals the two NADPH-binding domains inside the dimeric MtMtr in different conformations. NMR-titration shows that the MtMtr-MtMrx-1 interaction is characterized by a fast exchange regime and critical residues involved in the protein-protein interaction were identified. Using NMR spectroscopy and docking studies, the epitopes of MtMrx-1 and MtAhpE interaction are described, shedding new light into the interaction interface and mechanism of action. Finally, the essential residue of MtMrx-1 identified in the interaction with MtMtr and MtAhpE form a platform for structure-guided drug design against the versatile enzyme machinery of the mycothiol-dependent system inside M. tuberculosis.     

Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E_MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E_MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E_MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, MtbΔwhiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.     

Profiling of in vitro activities of urea-based inhibitors against cysteine synthases from Mycobacterium tuberculosis

CysK1 and CysK2 are two members of the cysteine/S-sulfocysteine synthase family in Mycobacterium tuberculosis, responsible for the de novo biosynthesis of l-cysteine, which is subsequently used as a building block for mycothiol. This metabolite is the first line defense of this pathogen against reactive oxygen and nitrogen species released by host macrophages after phagocytosis. In a previous medicinal chemistry campaign we had developed urea-based inhibitors of the cysteine synthase CysM with bactericidal activity against dormant M. tuberculosis. In this study we extended these efforts by examination of the in vitro activities of a library consisting of 71 urea compounds against CysK1 and CysK2. Binding was established by fluorescence spectroscopy and inhibition by enzyme assays. Several of the compounds inhibited these two cysteine synthases, with the most potent inhibitor displaying an IC₅₀ value of 2.5 µM for CysK1 and 6.6 µM for CysK2, respectively. Four of the identified molecules targeting CysK1 and CysK2 were also among the top ten inhibitors of CysM, suggesting that potent compounds could be developed with activity against all three enzymes.     

PEX5, the Shuttling Import Receptor for Peroxisomal Matrix Proteins,Is a Redox‐Sensitive Protein

Peroxisome maintenance depends on the import of nuclear‐encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C‐terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N‐terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox‐sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination‐deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.      

Redox Proteomic Analysis Reveals Oxidative Modifications of Proteins by Increased Levels of Intracellular Reactive Oxygen Species during Hypoxia Adaptation of Aspergillus fumigatus

Aspergillus fumigatus faces abrupt changes in oxygen concentrations at the site of infection. An increasing number of studies has demonstrated that elevated production of intracellular reactive oxygen species (ROS) under low oxygen conditions plays a regulatory role in modulating cellular responses for adaptation to hypoxia. To learn more about this process in A. fumigatus, intracellular ROS production during hypoxia has been determined. The results confirm increased amounts of intracellular ROS in A. fumigatus exposed to decreased oxygen levels. Moreover, nuclear accumulation of the major oxidative stress regulator AfYap1 is observed after low oxygen cultivation. For further analysis, iodoTMT labeling of redox‐sensitive cysteine residues is applied to identify proteins that are reversibly oxidized. This analysis reveals that proteins with important roles in maintaining redox balance and protein folding, such as the thioredoxin Asp f 29 and the disulfide‐isomerase PdiA, undergo substantial thiol modification under hypoxia. The data also show that the mitochondrial respiratory complex IV assembly protein Coa6 is significantly oxidized by hypoxic ROS. Deletion of the corresponding gene results in a complete absence of hypoxic growth, indicating the importance of complex IV during adaptation of A. fumigatus to oxygen‐limiting conditions.