Major Low Molecular Weight Thiol Research Articles

The Industrial Organism Corynebacterium glutamicum Requires Mycothiol as Antioxidant to Resist Against Oxidative Stress in Bioreactor Cultivations.

In aerobic environments, bacteria are exposed to reactive oxygen species (ROS). To avoid an excess of ROS, microorganisms are equipped with powerful enzymatic and non-enzymatic antioxidants. Corynebacterium glutamicum, a widely used industrial platform organism, uses mycothiol (MSH) as major low molecular weight (LMW) thiol and non-enzymatic antioxidant. In aerobic bioreactor cultivations, C. glutamicum becomes exposed to oxygen concentrations surpassing the air saturation, which are supposed to constitute a challenge for the intracellular MSH redox balance. In this study, the role of MSH was investigated at different oxygen levels (pO2) in bioreactor cultivations in C. glutamicum. Despite the presence of other highly efficient antioxidant systems, such as catalase, the MSH deficient ΔmshC mutant was impaired in growth in bioreactor experiments performed at pO2 values of 30%. At a pO2 level of 20%, this growth defect was abolished, indicating a high susceptibility of the MSH-deficient mutant towards elevated oxygen concentrations. Bioreactor experiments with C. glutamicum expressing the Mrx1-roGFP2 redox biosensor revealed a strong oxidative shift in the MSH redox potential (EMSH) at pO2 values above 20%. This indicates that the LMW thiol MSH is an essential antioxidant to maintain the robustness and industrial performance of C. glutamicum during aerobic fermentation processes.

Characterization and X-ray structure of the NADH-dependent coenzyme A disulfide reductase from Thermus thermophilus

The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.

Antimicrobial garlic-derived diallyl polysulfanes: Interactions with biological thiols in Bacillus subtilis

BackgroundDiallylpolysulfanes are the key constituents of garlic oils, known to exhibit broad spectrum anticancer and antimicrobial activity. Studies in vitro, and in mammalian cells, have shown they react, via thiol-polysulfane exchange, with their major low molecular weight thiol, glutathione. However, there are no detailed reports of diallylpolysulfane effects on other common thiol metabolites (cysteine and coenzyme A) or major thiol cofactors (e.g. bacillithiol) that many Gram positive bacteria produce instead of glutathione. MethodsDiallylpolysulfanes were individually purified then screened for antimicrobial activity against Bacillus subtilis. Their impact on thiol metabolites (bacillithiol, cysteine, coenzyme A, protein thiols allyl thiols//persulfides) in B. subtilis cultures were analysed, by HPLC. ResultsDiallylpolysulfane bioactivity increased with increasing chain length up to diallyltetrasulfane, but then plateaued. Within two minutes of treating B. subtilis with diallyltrisulfane or diallyltetrasulfane intracellular bacillithiol levels decreased by ~90%. Cysteine and CoA were also affected but to a lesser degree. This was accompanied by the accumulation of allyl thiol and allyl persulfide. A significant level of protein-S-allylation was also detected. ConclusionsIn addition to the major low molecular weight thiol, diallylpolysulfanes can also have an impact on other thiol metabolites and protein thiols. General significanceThis study shows the rapid parallel impact of polysulfanes on different biological thiols inside Bacillus subtilis alongside the concomitant generation of allyl thiols and persulfides.

Stable integration of the Mrx1-roGFP2 biosensor to monitor dynamic changes of the mycothiol redox potential in Corynebacterium glutamicum

Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (EMSH) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~− 296 mV. Consistent with its H2O2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal EMSH was observed in the sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level EMSH and the role of OxyR and KatA for efficient H2O2 detoxification under oxidative stress.

Changes in Glutathione Redox Potential Are Linked to Aβ42-Induced Neurotoxicity

Glutathione is the major low-molecular weight thiol ofeukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer's disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. Here, we combine genetically encoded redox sensors with our Drosophila models of amyloid-beta (Aβ) aggregation. We find that changes in glutathione redox potential (EGSH) closely correlate with disease onset and progression. We observe this redox imbalance specifically in neurons, but not in glia cells. EGSH changes and Aβ42 deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Aβ42-mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology.

The aldehyde dehydrogenase AldA contributes to the hypochlorite defense and is redox-controlled by protein S-bacillithiolation in Staphylococcus aureus

Staphylococcus aureus produces bacillithiol (BSH) as major low molecular weight (LMW) thiol which functions in thiol-protection and redox-regulation by protein S-bacillithiolation under hypochlorite stress. The aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus. Here, we have studied the expression, function, redox regulation and structural changes of AldA of S. aureus. Transcription of aldA was previously shown to be regulated by the alternative sigma factor SigmaB. Northern blot analysis revealed SigmaB-independent induction of aldA transcription under formaldehyde, methylglyoxal, diamide and NaOCl stress. Deletion of aldA resulted in a NaOCl-sensitive phenotype in survival assays, suggesting an important role of AldA in the NaOCl stress defense. Purified AldA showed broad substrate specificity for oxidation of several aldehydes, including formaldehyde, methylglyoxal, acetaldehyde and glycol aldehyde. Thus, AldA could be involved in detoxification of aldehyde substrates that are elevated under NaOCl stress. Kinetic activity assays revealed that AldA is irreversibly inhibited under H2O2 treatment in vitro due to overoxidation of Cys279 in the absence of BSH. Pre-treatment of AldA with BSH prior to H2O2 exposure resulted in reversible AldA inactivation due to S-bacillithiolation as revealed by activity assays and BSH-specific Western blot analysis. Using molecular docking and molecular dynamic simulation, we further show that BSH occupies two different positions in the AldA active site depending on the AldA activation state. In conclusion, we show here that AldA is an important target for S-bacillithiolation in S. aureus that is up-regulated under NaOCl stress and functions in protection under hypochlorite stress.

The glyceraldehyde-3-phosphate dehydrogenase GapDH of Corynebacterium diphtheriae is redox-controlled by protein S-mycothiolation under oxidative stress

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.

Monitoring global protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis under hypochlorite stress

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA anti-sigma factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.

Endothelial Cell Redox Regulation of Ischemic Angiogenesis.

The endothelium produces and responds to reactive oxygen and nitrogen species (RONS), providing important redox regulation to the cardiovascular system in physiology and disease. In no other situation are RONS more critical than in the response to tissue ischemia. Here, tissue healing requires growth factor-mediated angiogenesis that is in part dependent on low levels of RONS, which paradoxically must overcome the damaging effects of high levels of RONS generated as a result of ischemia. Although the generation of endothelial cell RONS in hypoxia/reoxygenation is acknowledged, the mechanism for their role in angiogenesis is still poorly understood. During ischemia, the major low molecular weight thiol glutathione (GSH) reacts with RONS and protein cysteines, producing GSH-protein adducts. Recent data indicate that GSH adducts on certain proteins are essential to growth factor responses in endothelial cells. Genetic deletion of the enzyme glutaredoxin-1, which selectively removes GSH protein adducts, improves, whereas its overexpression impairs revascularization of the ischemic hindlimb of mice. Ischemia-induced GSH adducts on specific cysteine residues of several proteins, including p65 NF-kB and the sarcoplasmic reticulum calcium ATPase 2, evidently promote ischemic angiogenesis. Identifying the specific proteins in the redox response to ischemia has provided therapeutic opportunities to improve clinical outcomes of ischemia.

Analysis of mutants disrupted in bacillithiol metabolism in Staphylococcus aureus

Bacillithiol (BSH), an α-anomeric glycoside of l-cysteinyl-d-glucosaminyl-l-malate, is a major low molecular weight thiol found in low GC Gram-positive bacteria, such as Staphylococcus aureus. Like other low molecular weight thiols, BSH is likely involved in protection against a number of stresses. We examined S. aureus transposon mutants disrupted in each of the three genes associated with BSH biosynthesis. These mutants are sensitive to alkylating stress, oxidative stress, and metal stress indicating that BSH and BSH-dependent enzymes are involved in protection of S. aureus. We further demonstrate that BshB, a deacetylase involved in the second step of BSH biosynthesis, also acts as a BSH conjugate amidase and identify S. aureus USA 300 LAC 2626 as a BSH-S-transferase, which is able to conjugate chlorodinitrobenzene, cerulenin, and rifamycin to BSH.

The Orphan Protein Bis-γ-glutamylcystine Reductase Joins the Pyridine Nucleotide Disulfide Reductase Family

Facile DNA sequencing became possible decades after many enzymes had been purified and characterized. Consequently, there are still "orphan" enyzmes for which activities are known but for which encoding genes have not been identified. Identification of the genes encoding orphan enzymes is important because it allows correct annotation of genes of unknown function or with misassigned function. Bis-γ-glutamylcystine reductase (GCR) is an orphan protein that was purified in 1988. This enzyme catalyzes the reduction of bis-γ-glutamylcystine. γ-Glutamylcysteine is the major low-molecular weight thiol in halobacteria. We purified GCR from Halobacterium sp. NRC-1 and identified the sequence of 23 tryptic peptides by nano-liquid chromatography electrospray ionization tandem mass spectrometry. These peptides cover 62% of the protein predicted to be encoded by a gene in Halobacterium sp. NRC-1 that is annotated as mercuric reductase. GCR and mercuric reductase activities were assayed using enzyme that was expressed in Escherichia coli and refolded from inclusion bodies. The enzyme had robust GCR activity but no mercuric reductase activity. The genomes of most, but not all, halobacteria for which whole genome sequences are available have close homologues of GCR, suggesting that there is more to be learned about the low-molecular weight thiols used in halobacteria.

Distribution and infection-related functions of bacillithiol in Staphylococcus aureus

Bacillithiol (Cys-GlcN-malate, BSH) serves as a major low molecular weight thiol in low GC Gram-positive bacteria including Bacillus species and a variety of Staphylococcus aureus strains. These bacteria do not produce glutathione (GSH). In this study, HPLC analyses were used to determine BSH levels in different S. aureus strains. Furthermore, the role of BSH in the resistance against oxidants and antibiotics and its function in virulence was investigated. We and others (Newton, G.L., Fahey, R.C., Rawat, M., 2012. Microbiology 158, 1117–1126) found that BSH is not produced by members of the S. aureus NCTC8325 lineage, such as strains 8325-4 and SH1000. Using bioinformatics we show that the BSH-biosynthetic gene bshC is disrupted by an 8-bp duplication in S. aureus NCTC8325. The functional bshC-gene from BSH-producing S. aureus Newman (NWMN_1087) was expressed in S. aureus 8325-4 to reconstitute BSH-synthesis. Comparison of the BSH-producing and BSH-minus strains revealed higher resistance of the BSH-producing strain against the antibiotic fosfomycin and the oxidant hypochlorite but not against hydrogen peroxide or diamide. In addition, a higher bacterial load of the BSH-producing strain was detected in human upper-airway epithelial cells and murine macrophages. This indicates a potential role of BSH in protection of S. aureus during infection.

Bacterial mechanisms of reversible protein S‐thiolation: structural and mechanistic insights into mycoredoxins

Mycobacteria produce millimolar concentrations of mycothiol (MSH) as their major low molecular weight thiol redox buffer. MSH-deficient mutants display increased sensitivity towards reactive oxygen, nitrogen and electrophilic species as well as alkylating agents and antibiotics. MSH is maintained in its reduced thiol state by the NADPH-dependent mycothiol disulphide reductase (Mtr). However, the redoxin that uses the MSH/Mtr/NADPH pathway for reduction of MSH-mixed protein disulphides, formed during oxidative stress, has long remained unknown. In this issue, Van Laer et al. report that MSH provides the reducing power for mycoredoxin-1 (Mrx1) in reduction of synthetic MSH-mixed disulphides. The reduced (dithiol) and oxidized (disulphide) solution structures of Mrx1 have been solved by nuclear magnetic resonance (NMR) spectroscopy. NMR time course experiments have also demonstrated the transient S-mycothiolation of the active site Cys14 of oxidized Mrx1 during reduction by the MSH/Mtr/NADPH electron pathway. The paper opens a new era of research to identify S-mycothiolated Mrx1 substrates and the function of MSH in redox regulation and virulence in Mycobacterium tuberculosis.

Examination of Mechanism of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Reveals Unexpected Role for Dynamic Tyrosine

Actinomycetes are a group of gram-positive bacteria that includes pathogenic mycobacterial species, such as Mycobacterium tuberculosis. These organisms do not have glutathione and instead utilize the small molecule mycothiol (MSH) as their primary reducing agent and for the detoxification of xenobiotics. Due to these important functions, enzymes involved in MSH biosynthesis and MSH-dependent detoxification are targets for drug development. The metal-dependent deacetylase N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) catalyzes the hydrolysis of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside to form 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside and acetate in MSH biosynthesis. Herein we examine the chemical mechanism of MshB. We demonstrate that the side chains of Asp-15, Tyr-142, His-144, and Asp-146 are important for catalytic activity. We show that NaF is an uncompetitive inhibitor of MshB, consistent with a metal-water/hydroxide functioning as the reactive nucleophile in the catalytic mechanism. We have previously shown that MshB activity has a bell-shaped dependence on pH with pK(a) values of ∼7.3 and 10.5 (Huang, X., Kocabas, E. and Hernick, M. (2011) J. Biol. Chem. 286, 20275-20282). Mutagenesis experiments indicate that the observed pK(a) values reflect ionization of Asp-15 and Tyr-142, respectively. Together, findings from our studies suggest that MshB functions through a general acid-base pair mechanism with the side chain of Asp-15 functioning as the general base catalyst and His-144 serving as the general acid catalyst, whereas the side chain of Tyr-142 probably assists in polarizing substrate/stabilizing the oxyanion intermediate. Additionally, our results indicate that Tyr-142 is a dynamic side chain that plays key roles in catalysis, modulating substrate binding, chemistry, and product release.

ChemInform Abstract: Mycothiol: Synthesis, Biosynthesis and Biological Functions of the Major Low Molecular Weight Thiol in Actinomycetes

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis [Nicely, N. I. , Parsonage, D., Paige, C., Newton, G. L., Fahey, R. C., Leonardi, R., Jackowski, S., Mallett, T. C., and Claiborne, A. (2007) Biochemistry 46, 3234-3245], and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 A resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 A resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367,425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.

Mycothiol: synthesis, biosynthesis and biological functions of the major low molecular weight thiol in actinomycetes

Actinomycetes produce mycothiol as their major low molecular weight thiol, which parallels the functions of glutathione found in prokaryotes and most Gram-negative bacteria. This review covers progress that has so far been made in terms of its distribution, biosynthesis and metabolic functions, as well as chemical syntheses of mycothiol and alternative substrates and inhibitors of mycothiol biosynthesis and mycothiol-dependent enzymes. 152 references are cited.

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology,

Coenzyme A (CoASH) is the major low-molecular weight thiol in Staphylococcus aureus and a number of other bacteria; the crystal structure of the S. aureus coenzyme A-disulfide reductase (CoADR), which maintains the reduced intracellular state of CoASH, has recently been reported [Mallett, T.C., Wallen, J.R., Karplus, P.A., Sakai, H., Tsukihara, T., and Claiborne, A. (2006) Biochemistry 45, 11278-89]. In this report we demonstrate that CoASH is the major thiol in Bacillus anthracis; a bioinformatics analysis indicates that three of the four proteins responsible for the conversion of pantothenate (Pan) to CoASH in Escherichia coli are conserved in B. anthracis. In contrast, a novel type III pantothenate kinase (PanK) catalyzes the first committed step in the biosynthetic pathway in B. anthracis; unlike the E. coli type I PanK, this enzyme is not subject to feedback inhibition by CoASH. The crystal structure of B. anthracis PanK (BaPanK), solved using multiwavelength anomalous dispersion data and refined at a resolution of 2.0 A, demonstrates that BaPanK is a new member of the Acetate and Sugar Kinase/Hsc70/Actin (ASKHA) superfamily. The Pan and ATP substrates have been modeled into the active-site cleft; in addition to providing a clear rationale for the absence of CoASH inhibition, analysis of the Pan-binding pocket has led to the development of two new structure-based motifs (the PAN and INTERFACE motifs). Our analyses also suggest that the type III PanK in the spore-forming B. anthracis plays an essential role in the novel thiol/disulfide redox biology of this category A biodefense pathogen.

Structure of Coenzyme A−Disulfide Reductase from Staphylococcus aureus at 1.54 Å Resolution,

Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Staphylococcus aureus; it is maintained in the reduced state by coenzyme A-disulfide reductase (CoADR), a homodimeric enzyme similar to NADH peroxidase but containing a novel Cys43-SSCoA redox center. The crystal structure of S. aureus CoADR has been solved using multiwavelength anomalous dispersion data and refined at a resolution of 1.54 A. The resulting electron density maps define the Cys43-SSCoA disulfide conformation, with Cys43-S(gamma) located at the flavin si face, 3.2 A from FAD-C4aF, and the CoAS- moiety lying in an extended conformation within a cleft at the dimer interface. A well-ordered chloride ion is positioned adjacent to the Cys43-SSCoA disulfide and receives a hydrogen bond from Tyr361'-OH of the complementary subunit, suggesting a role for Tyr361' as an acid-base catalyst during the reduction of CoAS-disulfide. Tyr419'-OH is located 3.2 A from Tyr361'-OH as well and, based on its conservation in known functional CoADRs, also appears to be important for activity. Identification of residues involved in recognition of the CoAS-disulfide substrate and in formation and stabilization of the Cys43-SSCoA redox center has allowed development of a CoAS-binding motif. Bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/disulfide homeostasis.

Purification and characterization of Mycobacterium tuberculosis 1 d- myo-inosityl-2-acetamido-2-deoxy-α- d-glucopyranoside deacetylase, MshB, a mycothiol biosynthetic enzyme

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.