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Robert C. Fahey - One of the best experts on this subject based on the ideXlab platform.
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the dinb superfamily includes novel Mycothiol bacillithiol and glutathione s transferases
Biochemistry, 2011Co-Authors: Gerald L. Newton, Mamta Rawat, Stephan S Leung, Judy I Wakabayashi, Robert C. FaheyAbstract:The superfamily of glutathione S-transferases has been the subject of extensive study but Actinobacteria produce Mycothiol (MSH) in place of glutathione and no Mycothiol S-transferase (MST) has been identified. Using Mycothiol and monochlorobimane as substrates a MST activity was detected in extracts of Mycobacterium smegmatis and purified sufficiently to allow identification of MSMEG_0887, a member the DUF664 family of the DinB superfamily, as the MST. The identity of the M. smegmatis and homologous Mycobacterium tuberculosis (Rv0443) enzymes was confirmed by cloning and the expressed proteins were found to be active with MSH but not bacillithiol (BSH) or glutathione (GSH). Bacillus subtilis YfiT is another member of the DinB superfamily but this bacterium produces BSH. The YfiT protein was shown to have Stransferase activity with monochlorobimane when assayed with BSH but not with MSH or GSH. Enterococcus faecalis EF_3021 shares some homology with MSMEG_0887 but this organism produces GSH but not MSH or BSH. Cloned and expressed EF_0321 was active with monochlorobimane and GSH but not with MSH or BSH. MDMPI_2 is another member of the DinB superfamily and has been previously shown to have Mycothiol-dependent maleylpyruvate isomerase activity. Three of the eight families of the DinB superfamily include proteins shown to catalyze thiol-dependent metabolic or detoxification activities. Since more than two-thirds of the sequences assigned to the DinB superfamily are members of these families it seems likely that such activity is dominant in the DinB superfamily.
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evaluation of ntf1836 as an inhibitor of the Mycothiol biosynthetic enzyme mshc in growing and non replicating mycobacterium tuberculosis
Bioorganic & Medicinal Chemistry, 2011Co-Authors: Gerald L. Newton, Nancy Buchmeier, James J La Clair, Robert C. FaheyAbstract:The Mycothiol biosynthesis enzyme MshC catalyzes the ligation of cysteine with the pseudodisaccharide GlcN-Ins and has been identified as an essential enzyme in Mycobacterium tuberculosis. We now report on the development of NTF1836 as a micromolar inhibitor of MshC. Using commercial libraries, we conducted preliminary structure-activity relationship (SAR) studies on NTF1836. Based on this data, NTF1836 and five structurally related compounds showed similar activity towards clinical strains of M. tuberculosis. A gram scale synthesis was developed to provide ample material for biological studies. Using this material, we determined that inhibition of M. tuberculosis growth by NTF1836 was accompanied by a fall in Mycothiol and an increase in GlcN-Ins consistent with the targeting of MshC. We also determined that NTF1836 kills non-replicating M. tuberculosis in the carbon starvation model of latency.
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bacillithiol is an antioxidant thiol produced in bacilli
Nature Chemical Biology, 2009Co-Authors: Gerald L. Newton, Mamta Rawat, Vishnu Karthik Jothivasan, Chris J Hamilton, James J La Clair, Tanya Budiarto, Al Claiborne, John D Helmann, Robert C. FaheyAbstract:Glutathione is a nearly ubiquitous, low-molecular-mass thiol and antioxidant, but it is conspicuously absent from most Gram-positive bacteria. We identify here the structure of bacillithiol, a newly described and abundant thiol produced by Bacillus species, Staphylococcus aureus and Deinococcus radiodurans. Bacillithiol is the α-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid and most probably functions as an antioxidant. Bacillithiol, like the structurally similar Mycothiol, may serve as a substitute for glutathione.
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An N-acyl homolog of Mycothiol is produced in marine actinomycetes
Archives of Microbiology, 2008Co-Authors: Gerald L. Newton, Paul R. Jensen, John B. Macmillan, William Fenical, Robert C. FaheyAbstract:Marine actinomycetes have generated much recent interest as a potentially valuable source of novel antibiotics. Like terrestrial actinomycetes the marine actinomycetes are shown here to produce Mycothiol as their protective thiol. However, a novel thiol, U25, was produced by MAR2 strain CNQ703 upon progression into stationary phase when secondary metabolite production occurred and became the dominant thiol. MSH and U25 were maintained in a reduced state during early stationary phase, but become significantly oxidized after 10 days in culture. Isolation and structural analysis of the monobromobimane derivative identified U25 as a homolog of Mycothiol in which the acetyl group attached to the nitrogen of cysteine is replaced by a propionyl residue. This N -propionyl-desacetyl-Mycothiol was present in 13 of the 17 strains of marine actinomycetes examined, including five strains of Salinispora and representatives of the MAR2, MAR3, MAR4 and MAR6 groups. Mycothiol and its precursor, the pseudodisaccharide 1- O -(2-amino-2-deoxy-α- d -glucopyranosyl)- d - myo -inositol, were found in all strains. High levels of Mycothiol S -conjugate amidase activity, a key enzyme in Mycothiol-dependent detoxification, were found in most strains. The results demonstrate that major thiol/disulfide changes accompany secondary metabolite production and suggest that Mycothiol-dependent detoxification is important at this developmental stage.
Gerald L. Newton - One of the best experts on this subject based on the ideXlab platform.
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the dinb superfamily includes novel Mycothiol bacillithiol and glutathione s transferases
Biochemistry, 2011Co-Authors: Gerald L. Newton, Mamta Rawat, Stephan S Leung, Judy I Wakabayashi, Robert C. FaheyAbstract:The superfamily of glutathione S-transferases has been the subject of extensive study but Actinobacteria produce Mycothiol (MSH) in place of glutathione and no Mycothiol S-transferase (MST) has been identified. Using Mycothiol and monochlorobimane as substrates a MST activity was detected in extracts of Mycobacterium smegmatis and purified sufficiently to allow identification of MSMEG_0887, a member the DUF664 family of the DinB superfamily, as the MST. The identity of the M. smegmatis and homologous Mycobacterium tuberculosis (Rv0443) enzymes was confirmed by cloning and the expressed proteins were found to be active with MSH but not bacillithiol (BSH) or glutathione (GSH). Bacillus subtilis YfiT is another member of the DinB superfamily but this bacterium produces BSH. The YfiT protein was shown to have Stransferase activity with monochlorobimane when assayed with BSH but not with MSH or GSH. Enterococcus faecalis EF_3021 shares some homology with MSMEG_0887 but this organism produces GSH but not MSH or BSH. Cloned and expressed EF_0321 was active with monochlorobimane and GSH but not with MSH or BSH. MDMPI_2 is another member of the DinB superfamily and has been previously shown to have Mycothiol-dependent maleylpyruvate isomerase activity. Three of the eight families of the DinB superfamily include proteins shown to catalyze thiol-dependent metabolic or detoxification activities. Since more than two-thirds of the sequences assigned to the DinB superfamily are members of these families it seems likely that such activity is dominant in the DinB superfamily.
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evaluation of ntf1836 as an inhibitor of the Mycothiol biosynthetic enzyme mshc in growing and non replicating mycobacterium tuberculosis
Bioorganic & Medicinal Chemistry, 2011Co-Authors: Gerald L. Newton, Nancy Buchmeier, James J La Clair, Robert C. FaheyAbstract:The Mycothiol biosynthesis enzyme MshC catalyzes the ligation of cysteine with the pseudodisaccharide GlcN-Ins and has been identified as an essential enzyme in Mycobacterium tuberculosis. We now report on the development of NTF1836 as a micromolar inhibitor of MshC. Using commercial libraries, we conducted preliminary structure-activity relationship (SAR) studies on NTF1836. Based on this data, NTF1836 and five structurally related compounds showed similar activity towards clinical strains of M. tuberculosis. A gram scale synthesis was developed to provide ample material for biological studies. Using this material, we determined that inhibition of M. tuberculosis growth by NTF1836 was accompanied by a fall in Mycothiol and an increase in GlcN-Ins consistent with the targeting of MshC. We also determined that NTF1836 kills non-replicating M. tuberculosis in the carbon starvation model of latency.
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bacillithiol is an antioxidant thiol produced in bacilli
Nature Chemical Biology, 2009Co-Authors: Gerald L. Newton, Mamta Rawat, Vishnu Karthik Jothivasan, Chris J Hamilton, James J La Clair, Tanya Budiarto, Al Claiborne, John D Helmann, Robert C. FaheyAbstract:Glutathione is a nearly ubiquitous, low-molecular-mass thiol and antioxidant, but it is conspicuously absent from most Gram-positive bacteria. We identify here the structure of bacillithiol, a newly described and abundant thiol produced by Bacillus species, Staphylococcus aureus and Deinococcus radiodurans. Bacillithiol is the α-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid and most probably functions as an antioxidant. Bacillithiol, like the structurally similar Mycothiol, may serve as a substitute for glutathione.
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An N-acyl homolog of Mycothiol is produced in marine actinomycetes
Archives of Microbiology, 2008Co-Authors: Gerald L. Newton, Paul R. Jensen, John B. Macmillan, William Fenical, Robert C. FaheyAbstract:Marine actinomycetes have generated much recent interest as a potentially valuable source of novel antibiotics. Like terrestrial actinomycetes the marine actinomycetes are shown here to produce Mycothiol as their protective thiol. However, a novel thiol, U25, was produced by MAR2 strain CNQ703 upon progression into stationary phase when secondary metabolite production occurred and became the dominant thiol. MSH and U25 were maintained in a reduced state during early stationary phase, but become significantly oxidized after 10 days in culture. Isolation and structural analysis of the monobromobimane derivative identified U25 as a homolog of Mycothiol in which the acetyl group attached to the nitrogen of cysteine is replaced by a propionyl residue. This N -propionyl-desacetyl-Mycothiol was present in 13 of the 17 strains of marine actinomycetes examined, including five strains of Salinispora and representatives of the MAR2, MAR3, MAR4 and MAR6 groups. Mycothiol and its precursor, the pseudodisaccharide 1- O -(2-amino-2-deoxy-α- d -glucopyranosyl)- d - myo -inositol, were found in all strains. High levels of Mycothiol S -conjugate amidase activity, a key enzyme in Mycothiol-dependent detoxification, were found in most strains. The results demonstrate that major thiol/disulfide changes accompany secondary metabolite production and suggest that Mycothiol-dependent detoxification is important at this developmental stage.
Yossef Avgay - One of the best experts on this subject based on the ideXlab platform.
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comparative analysis of mutants in the Mycothiol biosynthesis pathway in mycobacterium smegmatis
Biochemical and Biophysical Research Communications, 2007Co-Authors: Mamta Rawat, Chantale Johnson, Vanessa Cadiz, Yossef AvgayAbstract:The role of Mycothiol in mycobacteria was examined by comparative analysis of mutants disrupted in the four known genes encoding the protein machinery needed for Mycothiol biosynthesis. These mutants were sensitive to acid stress, antibiotic stress, alkylating stress, and oxidative stress indicating that Mycothiol and Mycothiol-dependent enzymes protect the mycobacterial cell against attack from various different types of stresses and toxic agents.
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innate protection of mycobacterium smegmatis against the antimicrobial activity of nitric oxide is provided by Mycothiol
Antimicrobial Agents and Chemotherapy, 2007Co-Authors: Chris Miller, Mamta Rawat, Todd Johnson, Yossef AvgayAbstract:Nitric oxide (NO) is an efficient antimicrobial agent. A role for Mycothiol in protecting mycobacteria from nitrosative damage was revealed by showing that a Mycobacterium smegmatis mutant is sensitive to NO. A direct correlation between NO and Mycothiol levels confirmed that Mycothiol is important for protecting mycobacteria from NO attack.
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Mycothiol dependent proteins in actinomycetes
Fems Microbiology Reviews, 2007Co-Authors: Mamta Rawat, Yossef AvgayAbstract:The pseudodisaccharide Mycothiol is present in millimolar levels as the dominant thiol in most species of Actinomycetales. The primary role of Mycothiol is to maintain the intracellular redox homeostasis. As such, it acts as an electron acceptor/donor and serves as a cofactor in detoxification reactions for alkylating agents, free radicals and xenobiotics. In addition, like glutathione, Mycothiol may be involved in catabolic processes with an essential role for growth on recalcitrant chemicals such as aromatic compounds. Following a little over a decade of research since the discovery of Mycothiol in 1994, we summarize the current knowledge about the role of Mycothiol as an enzyme cofactor and consider possible Mycothiol-dependent enzymes.
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purification and characterization of mycobacterium tuberculosis 1d myo inosityl 2 acetamido 2 deoxy α d glucopyranoside deacetylase mshb a Mycothiol biosynthetic enzyme
Protein Expression and Purification, 2006Co-Authors: Gerald L. Newton, Yossef Avgay, Robert C. FaheyAbstract:Abstract Mycothiol (MSH, AcCys-GlcN-Ins) is the major low molecular weight thiol in actinomycetes and is essential for growth of Mycobacterium tuberculosis . MshB, the GlcNAc-Ins deacetylase, is a key enzyme in MSH biosynthesis. MshB from M. tuberculosis was cloned, expressed, purified, and its properties characterized. Values of k cat and K m for MshB were determined for the biological substrate, GlcNAc-Ins, and several other good substrates. The substrate specificity of MshB was compared to that of M. tuberculosis Mycothiol S -conjugate amidase (Mca), a homologous enzyme having weak GlcNAc-Ins deacetylase activity. Both enzymes are metalloamidases with overlapping amidase activity toward Mycothiol S -conjugates (AcCySR-GlcN-Ins). The Ins residue and hydrophobic R groups enhance the activity with both MshB and Mca, but changes in the acyl group attached to GlcN have opposite effects on the two enzymes.
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Mycothiol dependent mycobacterial response to oxidative stress
FEBS Letters, 2006Co-Authors: Korine S E Ung, Yossef AvgayAbstract: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.
Joris Messens - One of the best experts on this subject based on the ideXlab platform.
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chemistry and redox biology of Mycothiol
Antioxidants & Redox Signaling, 2017Co-Authors: Aníbal M. Reyes, Mariaarmineh Tossounian, Brandan Pedre, Joris Messens, Rafael Radi, Maria Ines De Armas, Madia TrujilloAbstract:Abstract Significance: Mycothiol (MSH, AcCys-GlcN-Ins) is the main low-molecular weight (LMW) thiol of most Actinomycetes, including the human pathogen Mycobacterium tuberculosis that affects millions of people worldwide. Strains with decreased MSH content show increased susceptibilities to hydroperoxides and electrophilic compounds. In M. tuberculosis, MSH modulates the response to several antituberculosis drugs. Enzymatic routes involving MSH could provide clues for specific drug design. Recent Advances: Physicochemical data argue against a rapid, nonenzymatic reaction of MSH with oxidants, disulfides, or electrophiles. Moreover, exposure of the bacteria to high concentrations of two-electron oxidants resulted in protein Mycothiolation. The recently described glutaredoxin-like protein mycoredoxin-1 (Mrx-1) provides a route for catalytic reduction of Mycothiolated proteins, protecting critical cysteines from irreversible oxidation. The description of MSH/Mrx-1-dependent activities of peroxidases helped t...
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thiol redox and pka properties of Mycothiol the predominant low molecular weight thiol cofactor in the actinomycetes
ChemBioChem, 2016Co-Authors: Sunil Sharma, Joris Messens, Koen Van Laer, Chris J HamiltonAbstract:The thiol pKa and standard redox potential of Mycothiol, the major low-molecular-weight thiol cofactor in the actinomycetes, are reported. The measured standard redox potential reveals substantial discrepancies in one or more of the other previously measured intracellular parameters that are relevant to Mycothiol redox biochemistry.
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the corynebacterium glutamicum Mycothiol peroxidase is a reactive oxygen species scavenging enzyme that shows promiscuity in thiol redox control
Molecular Microbiology, 2015Co-Authors: Brandan Pedre, Khadija Wahni, Huriye Erdogan, Didier Vertommen, Luis M Mateos, Inge Van Molle, Almudena F Villadangos, Lucia Turell, Joris MessensAbstract: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 H2 O2 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 H2 O2 stress, and its gene expression is clearly induced upon H2 O2 challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.
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corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique Mycothiol redox relay mechanism
Journal of Biological Chemistry, 2015Co-Authors: Mariaarmineh Tossounian, Brandan Pedre, Khadija Wahni, Huriye Erdogan, Didier Vertommen, Inge Van Molle, Joris MessensAbstract: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.
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Mycothiol mycoredoxin 1 dependent reduction of the peroxiredoxin ahpe from mycobacterium tuberculosis
Journal of Biological Chemistry, 2014Co-Authors: Martín Hugo, Didier Vertommen, Joris Messens, Koen Van Laer, Aníbal M. Reyes, Rafael Radi, Madia TrujilloAbstract: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.
Brandan Pedre - One of the best experts on this subject based on the ideXlab platform.
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the antibacterial prodrug activator rv2466c is a Mycothiol dependent reductase in the oxidative stress response of mycobacterium tuberculosis
Journal of Biological Chemistry, 2017Co-Authors: Leonardo Astolfi Rosado, Brandan Pedre, Khadija Wahni, Giulia Degiacomi, David Young, Alfonso G De La Rubia, Francesca Boldrin, Edo Martens, Laura Marcospascual, Enea SanchovaelloAbstract:The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the Mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S-Mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel Mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it deMycothiolates and reduces a Mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free Mycothiol and Mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.
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chemistry and redox biology of Mycothiol
Antioxidants & Redox Signaling, 2017Co-Authors: Aníbal M. Reyes, Mariaarmineh Tossounian, Brandan Pedre, Joris Messens, Rafael Radi, Maria Ines De Armas, Madia TrujilloAbstract:Abstract Significance: Mycothiol (MSH, AcCys-GlcN-Ins) is the main low-molecular weight (LMW) thiol of most Actinomycetes, including the human pathogen Mycobacterium tuberculosis that affects millions of people worldwide. Strains with decreased MSH content show increased susceptibilities to hydroperoxides and electrophilic compounds. In M. tuberculosis, MSH modulates the response to several antituberculosis drugs. Enzymatic routes involving MSH could provide clues for specific drug design. Recent Advances: Physicochemical data argue against a rapid, nonenzymatic reaction of MSH with oxidants, disulfides, or electrophiles. Moreover, exposure of the bacteria to high concentrations of two-electron oxidants resulted in protein Mycothiolation. The recently described glutaredoxin-like protein mycoredoxin-1 (Mrx-1) provides a route for catalytic reduction of Mycothiolated proteins, protecting critical cysteines from irreversible oxidation. The description of MSH/Mrx-1-dependent activities of peroxidases helped t...
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the corynebacterium glutamicum Mycothiol peroxidase is a reactive oxygen species scavenging enzyme that shows promiscuity in thiol redox control
Molecular Microbiology, 2015Co-Authors: Brandan Pedre, Khadija Wahni, Huriye Erdogan, Didier Vertommen, Luis M Mateos, Inge Van Molle, Almudena F Villadangos, Lucia Turell, Joris MessensAbstract: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 H2 O2 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 H2 O2 stress, and its gene expression is clearly induced upon H2 O2 challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.
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corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique Mycothiol redox relay mechanism
Journal of Biological Chemistry, 2015Co-Authors: Mariaarmineh Tossounian, Brandan Pedre, Khadija Wahni, Huriye Erdogan, Didier Vertommen, Inge Van Molle, Joris MessensAbstract: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.
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corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique Mycothiol redox relay mechanism
Journal of Biological Chemistry, 2015Co-Authors: Mariaarmineh Tossounian, Brandan Pedre, Khadija Wahni, Huriye Erdogan, Didier Vertommen, Inge Van MolleAbstract: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. Background: Methionine sulfoxide post-translational modifications have an important new signaling role in cells. Results: Methionine sulfoxide reductase MsrA of the pathogenic actinomycete Corynebacterium diphtheriae (Cd-MsrA) uses a unique intramolecular redox relay mechanism coupled to Mycothiol. Conclusion: For methionine sulfoxide control, Cd-MsrA is flexible in receiving electrons from both the thioredoxin and the Mycothiol pathways. Significance: C. diphtheriae MsrA is a redox regulator for methionine sulfoxide signaling.
