The first step during bacillithiol (BSH) biosynthesis involves the formation of N ‐acetylglucosaminylmalate from UDP‐ N ‐acetylglucosamine and l ‐malate and is catalyzed by a GT4 class glycosyltransferase enzyme (BshA). Recombinant Staphylococcus aureus and Bacillus subtilis BshA were highly specific and active with l ‐malate but the former showed low activity with d ‐glyceric acid and the latter with d ‐malate. We show that BshA is inhibited by BSH and similarly that MshA (first enzyme of mycothiol biosynthesis) is inhibited by the final product MSH.

BACKGROUND:Oxygen is both essential and toxic to all forms of aerobic life and the chemical versatility and reactivity of thiols play a key role in both aspects. Cysteine thiol groups have key catalytic functions in enzymes but are readily damaged by reactive oxygen species (ROS). Low-molecular-weight thiols provide protective buffers against the hazards of ROS toxicity. Glutathione is the small protective thiol in nearly all eukaryotes but in prokaryotes the situation is far more complex. SCOPE OF REVIEW:This review provides an introduction to the diversity of low-molecular-weight thiol protective systems in bacteria. The topics covered include the limitations of cysteine as a protector, the multiple origins and distribution of glutathione biosynthesis, mycothiol biosynthesis and function in Actinobacteria, recent discoveries involving bacillithiol found in Firmicutes, new insights on the biosynthesis and distribution of ergothioneine, and the potential protective roles played by coenzyme A and other thiols. MAJOR CONCLUSIONS:Bacteria have evolved a diverse collection of low-molecular-weight protective thiols to deal with oxygen toxicity and environmental challenges. Our understanding of how many of these thiols are produced and utilized is still at an early stage. GENERAL SIGNIFICANCE:Extensive diversity existed among prokaryotes prior to evolution of the cyanobacteria and the development of an oxidizing atmosphere. Bacteria that managed to adapt to life under oxygen evolved, or acquired, the ability to produce a variety of small thiols for protection against the hazards of aerobic metabolism. Many pathogenic prokaryotes depend upon novel thiol protection systems that may provide targets for new antibacterial agents. This article is part of a Special Issue entitled Cellular functions of glutathione.

Bacillithiol (BSH), an α-anomeric glycoside of l-cysteinyl-d-glucosaminyl-l-malate, is a major low-molecular-mass thiol found in bacteria such as Bacillus sp., Staphylococcus aureus and Deinococcus radiodurans. Like other low-molecular-mass thiols such as glutathione and mycothiol, BSH is likely to be involved in protection against environmental toxins including thiol-reactive antibiotics. We report here a BSH-dependent detoxification mechanism in S. aureus. When S. aureus Newman strain was treated with monobromobimane and monochlorobimane, the cellular BSH was converted to the fluorescent S-conjugate BS-bimane. A bacillithiol conjugate amidase activity acted upon the BS-bimane to produce Cys-bimane, which was then acetylated by an N-acetyltransferase to generate N-acetyl-Cys-bimane, a mercapturic acid. An S. aureus mutant lacking BSH did not produce mercapturic acid when treated with monobromobimane and monochlorobimane, confirming the involvement of bacillithiol. Furthermore, treatment of S. aureus Newman with rifamycin, the parent compound of the first-line anti-tuberculosis drug, rifampicin, indicated that this thiol-reactive antibiotic is also detoxified in a BSH-dependent manner, since mercapturic acids of rifamycin were observed in the culture medium. These data indicate that toxins and thiol-reactive antibiotics are detoxified to less potent mercapturic acids in a BSH-dependent manner and then exported out of the cell in S. aureus.

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.

ABSTRACT The mshA ::Tn 5 mutant of Mycobacterium smegmatis does not produce mycothiol (MSH) and was found to markedly overproduce both ergothioneine and an ∼15-kDa protein determined to be organic hydroperoxide resistance protein (Ohr). An mshA ( G32D ) mutant lacking MSH overproduced ergothioneine but not Ohr. Comparison of the mutant phenotypes with those of the wild-type strain indicated the following: Ohr protects against organic hydroperoxide toxicity, whereas ergothioneine does not; an additional MSH-dependent organic hydroperoxide peroxidase exists; and elevated isoniazid resistance in the mutant is associated with both Ohr and the absence of MSH. Purified Ohr showed high activity with linoleic acid hydroperoxide, indicating lipid hydroperoxides as the likely physiologic targets. The reduction of oxidized Ohr by NADH was shown to be catalyzed by lipoamide dehydrogenase and either lipoamide or DlaT (SucB). Since free lipoamide and lipoic acid levels were shown to be undetectable in M. smegmatis , the bound lipoyl residues of DlaT are the likely source of the physiological dithiol reductant for Ohr. The pattern of occurrence of homologs of Ohr among bacteria suggests that the ohr gene has been distributed by lateral transfer. The finding of multiple Ohr homologs with various sequence identities in some bacterial genomes indicates that there may be multiple physiologic targets for Ohr proteins.

The superfamily of glutathione S-transferases has been the subject of extensive study; however, Actinobacteria produce mycothiol (MSH) in place of glutathione, and no mycothiol S-transferase (MST) has been identified. Using mycothiol and monochlorobimane as substrates, an 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 S-transferase activity with monochlorobimane when assayed with BSH but not with MSH or GSH. Enterococcus faecalis EF_3021 shares some homology with MSMEG_0887, but En. faecalis 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. Because 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.

CysQ is a 3′-phosphoadenosine-5′-phosphatase that dephosphorylates intermediates from the sulfate assimilation pathway of Mycobacterium tuberculosis (Mtb). Here, we demonstrate that cysQ disruption attenuates Mtb growth in vitro and decreases the biosynthesis of sulfated glycolipids but not major thiols, suggesting that the encoded enzyme specifically regulates mycobacterial sulfation.

Bacillithiol (BSH), the α-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid, is a major low-molecular-weight thiol in Bacillus subtilis and related bacteria. Here, we identify genes required for BSH biosynthesis and provide evidence that the synthetic pathway has similarities to that established for the related thiol (mycothiol) in the Actinobacteria. Consistent with a key role for BSH in detoxification of electrophiles, the BshA glycosyltransferase and BshB1 deacetylase are encoded in an operon with methylglyoxal synthase. BshB1 is partially redundant in function with BshB2, a deacetylase of the LmbE family. Phylogenomic profiling identified a conserved unknown function protein (COG4365) as a candidate cysteine-adding enzyme (BshC) that co-occurs in genomes also encoding BshA, BshB1, and BshB2. Additional evolutionarily linked proteins include a thioredoxin reductase homolog and two thiol:disulfide oxidoreductases of the DUF1094 (CxC motif) family. Mutants lacking BshA, BshC, or both BshB1 and BshB2 are devoid of BSH. BSH is at least partially redundant in function with other low-molecular-weight thiols: redox proteomics indicates that protein thiols are largely reduced even in the absence of BSH. At the transcriptional level, the induction of genes controlled by two thiol-based regulators (OhrR, Spx) occurs normally. However, BSH null cells are significantly altered in acid and salt resistance, sporulation, and resistance to electrophiles and thiol reactive compounds. Moreover, cells lacking BSH are highly sensitive to fosfomycin, an epoxide-containing antibiotic detoxified by FosB, a prototype for bacillithiol- S -transferase enzymes.

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 alpha-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.

Most Actinobacteria produce mycothiol as the major thiol. In addition to mycothiol Rhodococcus AD45 generates a substantial level of glutathione possibly using genes acquired in a lateral transfer. Instead of mycothiol, Rubrobacter radiotolerans and Rubrobacter xylanophilus produce glutathione, whose synthesis appears to involve enzymes substantially different from those in other organisms.

SUMMARY Mycothiol (MSH; AcCys-GlcN-Ins) is the major thiol found in Actinobacteria and has many of the functions of glutathione, which is the dominant thiol in other bacteria and eukaryotes but is absent in Actinobacteria . MSH functions as a protected reserve of cysteine and in the detoxification of alkylating agents, reactive oxygen and nitrogen species, and antibiotics. MSH also acts as a thiol buffer which is important in maintaining the highly reducing environment within the cell and protecting against disulfide stress. The pathway of MSH biosynthesis involves production of GlcNAc-Ins-P by MSH glycosyltransferase (MshA), dephosphorylation by the MSH phosphatase MshA2 (not yet identified), deacetylation by MshB to produce GlcN-Ins, linkage to Cys by the MSH ligase MshC, and acetylation by MSH synthase (MshD), yielding MSH. Studies of MSH mutants have shown that the MSH glycosyltransferase MshA and the MSH ligase MshC are required for MSH production, whereas mutants in the MSH deacetylase MshB and the acetyltransferase (MSH synthase) MshD produce some MSH and/or a closely related thiol. Current evidence indicates that MSH biosynthesis is controlled by transcriptional regulation mediated by σ B and σ R in Streptomyces coelicolor . Identified enzymes of MSH metabolism include mycothione reductase (disulfide reductase; Mtr), the S -nitrosomycothiol reductase MscR, the MSH S -conjugate amidase Mca, and an MSH-dependent maleylpyruvate isomerase. Mca cleaves MSH S -conjugates to generate mercapturic acids (AcCySR), excreted from the cell, and GlcN-Ins, used for resynthesis of MSH. The phenotypes of MSH-deficient mutants indicate the occurrence of one or more MSH-dependent S -transferases, peroxidases, and mycoredoxins, which are important targets for future studies. Current evidence suggests that several MSH biosynthetic and metabolic enzymes are potential targets for drugs against tuberculosis. The functions of MSH in antibiotic-producing streptomycetes and in bioremediation are areas for future study.

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-alpha-Dglucopyranosyl)-Dmyo-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.

Mycothiol (MSH) is the major thiol in Actinobacteria and plays a role analogous to that of glutathione. The biosynthetic pathway has been established in mycobacteria and is initiated by the glycosyltransferase MshA. A key mycothiol-dependent detoxification pathway utilizes the amidase (Mca) to cleave mycothiol S-conjugates to produce GlcN-Ins and a mercapturic acid excreted from the cell. How expression of mycothiol genes is regulated in mycobacteria has been unclear so the report in this issue by Park and Roe showing that in Streptomyces coelicolor the redox controlled anti-sigma factor RsrA that binds the regulator sigma(R) controls key elements of mycothiol metabolism is a major advance. Conditions that deplete thiols are shown to induce directly expression of sigR, rsrA, mshA and mca, as well as the thioredoxin reductase-thioredoxin system, generating an autoregulatory cycle that persists until the thiol-depleting condition is alleviated. Evidence for indirect induction of mshB-D to support mycothiol biosynthesis is also presented. It was shown in vitro that mycothiol, like reduced thioredoxin and dithiothreitol, can reduce oxidized RsrA to activate its binding to sigma(R). These studies establish for the first time how mycothiol metabolism is regulated to cope with stress from thiol reactive toxins.

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.

ABSTRACT Mycothiol ([MSH] AcCys-GlcN-Ins, where Ac is acetyl) is the major thiol produced by Mycobacterium smegmatis and other actinomycetes. Mutants deficient in MshA (strain 49) or MshC (transposon mutant Tn 1 ) of MSH biosynthesis produce no MSH. However, when stationary phase cultures of these mutants were incubated in medium containing MSH, they actively transported it to generate cellular levels of MSH comparable to or greater than the normal content of the wild-type strain. When these MSH-loaded mutants were transferred to MSH-free preconditioned medium, the cellular MSH was catabolized to generate GlcN-Ins and AcCys. The latter was rapidly converted to Cys by a high deacetylase activity assayed in extracts. The Cys could be converted to pyruvate by a cysteine desulfhydrase or used to regenerate MSH in cells with active MshC. Using MSH labeled with [U- 14 C]cysteine or with [6- 3 H]GlcN, it was shown that these residues are catabolized to generate radiolabeled products that are ultimately lost from the cell, indicating extensive catabolism via the glycolytic and Krebs cycle pathways. These findings, coupled with the fact the myo -inositol can serve as a sole carbon source for growth of M. smegmatis , indicate that MSH functions not only as a protective cofactor but also as a reservoir of readily available biosynthetic precursors and energy-generating metabolites potentially important under stress conditions. The half-life of MSH was determined in stationary phase cells to be ∼50 h in strains with active MshC and 16 ± 3 h in the MshC-deficient mutant, suggesting that MSH biosynthesis may be a suitable target for drugs to treat dormant tuberculosis.

Mycothiol is the major thiol produced by mycobacteria and is required for growth of Mycobacterium tuberculosis. The final three steps in the biosynthesis of mycothiol have been fully elucidated but the initial steps have been unclear. A glycosyltransferase, MshA, is required for production of the mycothiol precursor, 1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol, but its substrates and immediate products were unknown. In this study, we show that the N-acetylglucosamine donor is UDP-N-acetylglucosamine and that the N-acetylglucosamine acceptor is 1L-myo-inositol 1-phosphate. The reaction generates UDP and 1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol 3-phosphate. Using cell-free extracts of M. smegmatis mc(2)155, little activity was obtained with myo-inositol, 1D-myo-inositol 1-phosphate, or myo-inositol 2-phosphate as the N-acetylglucosamine acceptor. A phosphatase, designated MshA2, is required to dephosphorylate 1-O-(2-acetamido-2-deoxy-D-glucopyranosyl)-D- myo-inositol 3-phosphate to produce 1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo- inositol. The latter is deacetylated, ligated with cysteine, and the cysteinyl amino group acetylated by acetyl-CoA to complete the mycothiol biosynthesis pathway. Uptake and concentration of myo-[C-14] inositol is rapid in Mycobacterium smegmatis and leads to production of radio labeled inositol1-phosphate and my cothiol. This demonstrates the presence of a myo- inositol transporter and a kinase that generates 1L-myo-inositol1-phosphate. The biochemical pathway of mycothiol biosynthesis is now fully elucidated.