N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis

Mycothiol is a novel thiol produced only by actinomycetes and is the major low-molecular-weight thiol in mycobacteria. Mycothiol was previously shown to be synthesized from 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside by ligation with cysteine followed by acetylation. A novel mycothiol-dependent detoxification enzyme, mycothiol conjugate amidase, was recently identified in Mycobacterium smegmatis and shown to have a homolog, Rv1082, in Mycobacterium tuberculosis. In the present study we found that a protein encoded by the M. tuberculosis open reading frame Rv1170, a homolog of Rv1082, possesses weak mycothiol conjugate amidase activity but shows substantial deacetylation activity with 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins), a hypothetical mycothiol biosynthetic precursor. The availability of this protein enabled us to develop an assay for GlcNAc-Ins, which was used to demonstrate that GlcNAc-Ins is present in M. smegmatis at a level about twice that of mycothiol. It was shown that GlcNAc-Ins is absent in mycothiol-deficient mutant strain 49 of M. smegmatis and that this strain can concentrate GlcNAc-Ins from the medium and convert it to mycothiol. This demonstrates that GlcNAc-Ins is a key intermediate in the pathway of mycothiol biosynthesis. Assignment of Rv1170 as the gene coding the deacetylase in the M. tuberculosis genome represents the first identification of a gene of the mycothiol biosynthesis pathway. The presence of a large cellular pool of substrate for this enzyme suggests that it may be important in regulating mycothiol biosynthesis.     

The glycosyltransferase gene encoding the enzyme catalyzing the first step of mycothiol biosynthesis (mshA)

Mycothiol is the major thiol present in most actinomycetes and is produced from the pseudodisaccharide 1D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins). A transposon mutant of Mycobacterium smegmatis shown to be GlcNAc-Ins and mycothiol deficient was sequenced to identify a putative glycosyltransferase gene designated mshA. The ortholog in Mycobacterium tuberculosis, Rv0486, was used to complement the mutant phenotype.     

ATP-dependent L-cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase,mycothiol biosynthesis enzyme MshC,is related to class I cysteinyl-tRNA synthetases

Mycothiol is a novel thiol produced only by actinomycetes and is the major low molecular weight thiol in mycobacteria. The mycothiol biosynthetic pathway has been postulated to involve ATP-dependent ligation of L-cysteine (Cys) with 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside; GlcN-Ins) catalyzed by MshC to produce Cys-GlcN-Ins. The ligase activity was purified approximately 2400-fold from Mycobacterium smegmatis and two proteins of slightly different M(r) approximately 47000 were identified with MshC activity. The N-terminal sequence of the smaller protein revealed that it was coded by a gene in the databases for M. smegmatis and M. tuberculosis previously designated as cysS2. The larger protein was coded by the same gene in M. smegmatis but included an eight amino acid N-terminal extension involving a different start codon. The ligase was found to have K(m) values of 40 +/- 3 and 72 +/- 9 microM for Cys and GlcN-Ins, respectively. The cysS2 gene was thought to encode a second cysteinyl-tRNA synthetase in addition to cysS but the present results indicate that cysS2 is actually the mshC gene encoding ATP-dependent Cys:GlcN-Ins ligase.     

Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside and mycothiol biosynthesis

Mycothiol (MSH) is the major low molecular weight thiol in mycobacteria. Two chemical mutants with low MSH and one with no MSH (strain 49) were produced in Mycobacterium smegmatis mc2155 to assess the role of MSH in mycobacteria. Strain 49 was shown to not produce 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside (GlcN-Ins), an intermediate in MSH biosynthesis. Relative to the parent strain, mutant 49 formed colonies more slowly on solid media and was more sensitive to H2O2 and rifampin, but less sensitive to isoniazid. Complementation of mutant 49 with DNA from M. tuberculosis H37Rv partially restored production of GlcN-Ins and MSH, and resistance to H2O2, but largely restored colony growth rate and sensitivity to rifampin and isoniazid. The results indicate that MSH and GlcN-Ins are not essential for in vitro survival of mycobacteria but may play significant roles in determining the sensitivity of mycobacteria to environmental toxins.     

Characterization of Mycobacterium tuberculosis mycothiol S-conjugate amidase

Mycothiol is comprised of N-acetylcysteine (AcCys) amide linked to 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) and is the predominant thiol found in most actinomycetes. Mycothiol S-conjugate amidase (Mca) cleaves the amide bond of mycothiol S-conjugates of a variety of alkylating agents and xenobiotics, producing GlcN-Ins and a mercapturic acid that can be excreted from the cell. Mca of Mycobacterium tuberculosis (Rv1082) was cloned and expressed as a soluble protein in Escherichia coli. The protein contained 1.4 +/- 0.1 equiv of zinc after purification, indicating that Mca is a metalloprotein with zinc as the native metal. Kinetic studies of Mca activity with 14 substrates demonstrated that Mca is highly specific for the mycothiol moiety of mycothiol S-conjugates and relatively nonspecific for the structure of the sulfur-linked conjugate. The deacetylase activity of Mca with GlcNAc-Ins is small but significant and failed to saturate at up to 2 mM GlcNAc-Ins, indicating that Mca may contribute modestly to the production of GlcN-Ins when GlcNAc-Ins levels are high. The versatility of Mca can be seen in its ability to react with a broad range of mycothiol S-conjugates, including two different classes of antibiotics. The mycothiol S-conjugate of rifamycin S was produced under physiologically relevant conditions and was shown to be a substrate for Mca in both oxidized and reduced forms. Significant activity was also seen with the mycothiol S-conjugate of the antibiotic cerulenin as a substrate for Mca.     

Differential expression of mycothiol pathway genes: are they affected by antituberculosis drugs?

Mycothiol (MSH) is the major cellular thiol in Mycobacterium tuberculosis (M.tb). We hypothesize that the mycothiol-dependent detoxification pathway may serve an important role during oxygen stress management in M. tuberculosis, derived from normal aerobic metabolism, the macrophage environment and through the action of anti-tubercular antibiotics, such as Isoniazid (INH). Total mRNA and DNA were isolated from M. bovis BCG at different stages of growth in 7H9 mycobacterial medium. Three genes involved in mycothiol metabolism and encoding the enzymes mycothiol S-conjugate amidase (Mca, Rv1082), NADPH dependent mycothiol reductase (mtr, Rv2855), and N-Acetyl-1-D-myo-Inosityl-2-Amino-2-Deoxy-alpha-D-Glucopyranoside Deacetylase (GlcNAc-Ins deacetylase, Rv1170 or mshB) were investigated for genomic rearrangements and expression. The results show that the genomic domains of the genes remain conserved in evolutionary diverse and unrelated M. tuberculosis isolates. The genes encoding enzymes implicated in mycothiol reduction, mtr (Rv2855) and the mycothiol-dependant detoxification of electrophilic agents, Mca (Rv1082), are shown to be actively transcribed during logarithmic M. bovis BCG growth. The gene encoding GlcNAc-Ins deacetylase (the rate limiting mycothiol biosynthesis step) shows induction in the presence of INH. Antisense oligonucleotides to both GlcNAc-Ins deacetylase (Rv1170) and mtr (Rv2855) mRNA affect mycobacterial growth. In conclusion the results presented here suggest that these enzymes are sensitive to free radical generating antituberculosis drugs and may be useful targets for new drug development.     

The crystal structure of 1-D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) from Mycobacterium tuberculosis reveals a zinc hydrolase with a lactate dehydrogenase fold

Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two beta-sheets and two alpha-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD+ co-factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB.     

A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol S-conjugate amidase

Mycothiol, 1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (MSH), is composed of N-acetylcysteine (AcCys) amide linked to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes. When Mycobacterium smegmatis was treated with the alkylating agent monobromobimane (mBBr), the cellular mycothiol was converted to its bimane derivative (MSmB). The latter was rapidly cleaved to produce GlcN-Ins and the bimane derivative of N-acetylcysteine (AcCySmB), a mercapturic acid that was rapidly exported from the cells into the medium. The other product of cleavage, GlcN-Ins, was retained in the cell and utilized in the resynthesis of mycothiol. The mycothiol S-conjugate amidase (amidase) responsible for cleaving MSmB was purified to homogeneity from M. smegmatis. A value of K(m) = 95 +/- 8 microM and a value of k(cat) = 8 s(-)(1) was determined for the amidase with MSmB as substrate. Activity with 100 microM mycothiol or with the monobromobimane derivative of 1-D-myo-inosityl-2-(L-cysteinyl)amido-2-deoxy-alpha-D-glucopyra nos ide (CySmB-GlcN-Ins) or of 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-(alpha, beta)-D-glucopyranoside (AcCySmB-GlcN) was at least 10(3) lower than with 100 microM MSmB, demonstrating that the amidase is highly specific for S-conjugates of mycothiol. Conjugates of mycothiol with the antibiotic cerulenin, N-ethylmaleimide, 3-(N-maleimidopropionyl)-biocytin, and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin also exhibited significant activity. The sequence of the amino-terminal 20 residues was determined, and an open reading frame (Rv1082) coding for 288 residues having an identical predicted amino-terminal amino acid sequence was identified in the Mycobacterium tuberculosis genome. The Rv1082 gene (mca) from M. tuberculosis was cloned and expressed in Escherichia coli, and the expressed protein was shown to have substrate specificity similar to the amidase from M. smegmatis. These results indicate that mycothiol and mycothiol S-conjugate amidase play an important role in the detoxification of alkylating agents and antibiotics.     

Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes

Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn 5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1 D-myo-inosityl 2- L-cysteinylamido-2-deoxy-alpha-D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene.     

Mycothiol import by Mycobacterium smegmatis and function as a resource for metabolic precursors and energy production

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 Tn1) 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 approximately 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.     

Synthesis of 1-D- and 1-L-myo-inosityl 2-N-acetamido-2-deoxy-alpha-D-glucopyranoside establishes substrate specificity of the Mycobacterium tuberculosis enzyme AcGI deacetylase

Mycothiol (MSH, 1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside) is the principal low molecular weight thiol in actinomycetes. The enzyme 1-D-myo-inosityl 2-N-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase (AcGI deacetylase) is involved in the biosynthesis of MSH and forms the free amine 1-D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside, which is used in the third of four steps of MSH biosynthesis. Here, we report the synthesis of two isomers of AcGI, which contain either 1-L-myo-inositol or 1-D-myo-inositol. These synthetic products were used to investigate substrate specificity of the Mycobacterium tuberculosis enzyme AcGI deacetylase.     

Positional isotope exchange analysis of the Mycobacterium smegmatis cysteine ligase (MshC)

MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins, which is an intermediate in the biosynthetic pathway of mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (MSH or AcCys-GlcN-Ins). MSH is produced by Mycobacterium tuberculosis, members of the Actinomycetes family, to maintain an intracellular reducing environment and protect against oxidative and antibiotic induced stress. The biosynthesis of MSH is essential for cell growth, and therefore, the MSH biosynthetic enzymes present potential targets for inhibitor design. The formation of kinetically competent adenylated intermediates was suggested by the observation of positional isotope exchange (PIX) reaction using [betagamma-(18)O6]-ATP in the presence of cysteine. The PIX rate depends on the presence of cysteine and increases with concentrations of cysteine. The loss of PIX activity upon the addition of small concentrations of pyrophosphatase suggests that the PP(i) is free to dissociate from the active site of cysteine ligase into the bulk solution. The PIX activity is also eliminated at high concentrations of GlcN-Ins, consistent with the mechanism in which GlcN-Ins binds after cysteine-adenylate formation. This PIX analysis confirms that MshC catalyzes the formation of a kinetically competent cysteinyl-adenylate intermediate after the addition of ATP and cysteine.     

A mycothiol synthase mutant of Mycobacterium tuberculosis has an altered thiol-disulfide content and limited tolerance to stress

Mycothiol (MSH) (acetyl-Cys-GlcN-Ins) is the major low-molecular-mass thiol in Mycobacterium tuberculosis. MSH has antioxidant activity, can detoxify a variety of toxic compounds, and helps to maintain the reducing environment of the cell. The production of MSH provides a potential novel target for tuberculosis treatment. Biosynthesis of MSH requires at least four genes. To determine which of these genes is essential in M. tuberculosis, we have been constructing targeted gene disruptions. Disruption in the mshC gene is lethal to M. tuberculosis, while disruption in the mshB gene results in MSH levels 20 to 100% of those of the wild type. For this study, we have constructed a targeted gene disruption in the mshD gene that encodes mycothiol synthase, the final enzyme in MSH biosynthesis. The mshD mutant produced approximately 1% of normal MSH levels but high levels of the MshD substrate Cys-GlcN-Ins and the novel thiol N-formyl-Cys-GlcN-Ins. Although N-formyl-Cys-GlcN-Ins was maintained in a highly reduced state, Cys-GlcN-Ins was substantially oxidized. In both the wild type and the mshD mutant, cysteine was predominantly oxidized. The M. tuberculosis mshD mutant grew poorly on agar plates lacking catalase and oleic acid and in low-pH media and had heightened sensitivity to hydrogen peroxide. The inability of the mshD mutant to survive and grow in macrophages may be associated with its altered thiol-disulfide status. It appears that N-formyl-Cys-GlcN-Ins serves as a weak surrogate for MSH but is not sufficient to support normal growth of M. tuberculosis under stress conditions such as those found within the macrophage.     

Mycothiol biochemistry

Mycothiol (MSH) is a novel thiol comprised of N-acetylcysteine amide-linked to GlcN-alpha(1-1)-Ins. It is the major thiol in most actinomycetes and is produced at millimolar levels in mycobacteria and streptomycetes. MSH biosynthesis occurs by linkage of GlcNAc to Ins, deacetylation to GlcN-Ins, ligation of the latter to L-cysteine, and transacetylation of the cysteinyl residue by CoASAc to produce MSH. The genes encoding the respective enzymes have been designated mshA, mshB, mshC, and mshD; all but mshA have been identified. Mycobacterium smegmatis mutants deficient in mshA, mshC, and mshD have been characterized. MSH plays a significant role in the detoxification of thiol-reactive substances, including formaldehyde, various electrophiles, and antibiotics. Mycothiol S-conjugates derived from electrophiles and antibiotics are cleaved by mycothiol S-conjugate amidase to release GlcN-Ins, used to resynthesize MSH, and a mercapturic acid which is excreted from the cell. A mycothiol-disulfide-selective reductase has been identified and likely helps to maintain cellular MSH in the reduced state. Mycothiol biochemistry has characteristics similar to those of glutathione but also has a variety of unique features.     

Mycothiol is essential for growth of Mycobacterium tuberculosis Erdman

Mycothiol (MSH) is the major low-molecular-mass thiol in mycobacteria and is associated with the protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. The biosynthesis of MSH is a multistep process, with the enzymatic reaction designated MshC being the ligase step in MSH production. A targeted disruption of the native mshC gene in M. tuberculosis Erdman produced no viable clones possessing either a disrupted mshC gene or reduced levels of MSH. However, when a second copy of the mshC gene was incorporated into the chromosome prior to the targeted disruption, multiple clones having the native gene disrupted and the second copy of mshC intact were obtained. These clones produced normal levels of MSH. These results demonstrate that the mshC gene and, more generally, the production of MSH are essential for the growth of M. tuberculosis Erdman under laboratory conditions.     

Thiols in nitric oxide synthase-containing Nocardia sp. strain NRRL 5646

Mycothiol (MSH) [1-D-myo-inosityl-2-(N-acetyl-l-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside], isolated as the bimane derivative, was established to be the major thiol in Nocardia sp. strain NRRL 5646, a species most closely related to Nocardia brasiliensis strain DSM 43758(T). Thiol formation and detection of MSH-dependent formaldehyde dehydrogenase activity in cell extracts are relevant to the possible modulation of nitric oxide toxicity generated by strain NRRL 5646.     

Targeted mutagenesis of the Mycobacterium smegmatis mca gene, encoding a mycothiol-dependent detoxification protein

Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner.     

Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC)

Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-d-myo-inosityl-2-(N-acetyl-l-cysteinyl)amido-2-deoxy-alpha-d-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-MshD. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady-state and pre-steady-state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady-state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s-1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5'-O-[N-(l-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of approximately 306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of approximately 9.4 and approximately 5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.     

Regulation of mycothiol metabolism by sigma(R) and the thiol redox sensor anti-sigma factor RsrA

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.