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 cooccurs 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 epoxidecontaining antibiotic detoxified by FosB, a prototype for bacillithiol-Stransferase enzymes.Bacillus subtilis | glutathione | glutaredoxin | mycothiol | thioredoxin

Glutathione is a nearly ubiquitous low-molecular-weight thiol and antioxidant, although it is conspicuously absent from most Gram-positive bacteria. We identify here the structure of bacillithiol, a novel 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 likely functions as an antioxidant. Bacillithiol, like structurally similar mycothiol, may serve as a substitute for glutathione.

Development of new delivery systems that deliver the potential drug specifically to the target site in order to meet the therapeutic needs of the patients at the required time and level remains the key challenge in the field of pharmaceutical biotechnology. Developments in this context to achieve desired goal has led to the evolution of the multidisciplinary field nanobiotechnology which involves the combination of two most promising technologies of 21st century-biotechnology and nanotechnology. Nanobiotechnology encompasses a wide array of different techniques to improve the delivery of biotech drugs, and nanoparticles offer the most suitable form whose properties can be tailored by chemical methods. This review highlights the different types of nanoparticulate delivery systems employed for biotech drugs in the field of molecular medicine with a short overlook at its applications and the probable associated drawbacks.

Mycothiol (MSH; 1D-myo-inosityl 2-[N-acetyl-L-cysteinyl]amido-2-deoxy-␣-D-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc2 155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1D-myo-inosityl 2-deoxy-␣-D-glucopyranoside to levels 20-to 25-fold the level found in the parent strain. The cysteine:1D-myo-inosityl 2-amino-2-deoxy-␣-D-glucopyranoside ligase (MshC) activities of the three mutant strains were <2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics. Fig. 1A) is a low-molecular-weight thiol that serves functions in mycobacteria analogous to those of glutathione in gram-negative bacteria and eukaryotes (7). These functions include protection against oxidative damage and inactivation of electrophilic toxins (15,17). The distribution of MSH is limited to gram-positive actinomycetes, and of all the species tested, mycobacteria appear to generate the highest levels of MSH (14). The absence of MSH in mammalian cells suggests that the enzymes involved in the metabolism of MSH may be attractive drug targets for either rational drug design or screening of libraries of inhibitory compounds (25). Mycothiol (MSH;Although studies of MSH biosynthesis are still at an early stage, as shown in Fig. 1B, elements of the overall pathway have been described (1, 4, 16). Moreover, direct measurements of intermediates in bacterial extracts that support the proposed biosynthetic pathway have been reported (1). The genes for the MSH biosynthesis pathway were designated mshA, mshB, mshC, and mshD (16), with the corresponding enzymes labeled as shown in Fig. 1. It has been shown that 1D-myo-inosityl 2-acetamino-2-deoxy-␣-D-glucopyranoside (GlcNAc-Ins) is an intermediate (16), and we presume that its formation is the first dedicated step in MSH biosynthesis. Glucosaminylinositol is formed by deacetylation of GlcNAc-Ins. We have previously identified m...

SummaryTo survive hostile conditions, the bacterial pathogen Mycobacterium tuberculosis produces millimolar concentrations of mycothiol as a redox buffer against oxidative stress. The reductases that couple the reducing power of mycothiol to redox active proteins in the cell are not known. We report a novel mycothiol-dependent reductase (mycoredoxin-1) with a CGYC catalytic motif. With mycoredoxin-1 and mycothiol deletion strains of Mycobacterium smegmatis, we show that mycoredoxin-1 and mycothiol are involved in the protection against oxidative stress. Mycoredoxin-1 acts as an oxidoreductase exclusively linked to the mycothiol electron transfer pathway and it can reduce S-mycothiolated mixed disulphides. Moreover, we solved the solution structures of oxidized and reduced mycoredoxin-1, revealing a thioredoxin fold with a putative mycothiol-binding site.With HSQC snapshots during electron transport, we visualize the reduction of oxidized mycoredoxin-1 as a function of time and find that mycoredoxin-1 gets S-mycothiolated on its N-terminal nucleophilic cysteine. Mycoredoxin-1 has a redox potential of -218 mV and hydrogen bonding with neighbouring residues lowers the pK a of its N-terminal nucleophilic cysteine. Determination of the oxidized and reduced structures of mycoredoxin-1, better understanding of mycothiol-dependent reactions in general, will likely give new insights in how M. tuberculosis survives oxidative stress in human macrophages.

FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G + C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The Km values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn2+ and Mg2+ are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS– fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.

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.

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 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 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.