A Novel Mycothiol-Dependent Detoxification Pathway in Mycobacteria Involving Mycothiol S-Conjugate Amidase

Abstract

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.

Differential Expression of Mycothiol Pathway Genes: Are they Affected by Antituberculosis Drugs?

“…Our results suggest therefore that the control (activation) may be from up-regulation of transcription under conditions of demand. We also show that the enzymes responsible for the reduction (and therefore maintenance of the cellular redox state (32)) of MSH-disulfide (the oxidised form of mycothiol), mtr (32,33) is expressed in the logarithmic growth stage of M. bovis BCG as is Mca (12). This suggests that these enzymes may be constitutively expressed, which is in agreement with current information which suggests that these are not the control points for mycothiol synthesis.…”

“…Upon entry into the bacterial cell, mycothiol may bind electrophiles via a S-conjugate bond, which can be cleaved internally by a S-conjugate amidase to release the molecule in the form of a hydrophobic mercapturic acid from the cell (12). Recently the mycothiol specific S-conjugate amidase was cloned and shown to be important in the detoxification of electrophilic agents in M. smegmatis (12). These authors hypothesized that this enzyme may furthermore play a role in the adaptive response to and detoxification of antibiotics.…”

Glutathione disulfide and S‐nitrosoglutathione detoxification by Mycobacteriumtuberculosis thioredoxin system

“…GSSG (Sigma) and GSNO (Sigma) were assayed at 26 lM, while H 2 O 2 was used at a final concentration of 5 lM. A thiol-containing crude extract from M. smegmatis (6 mM as assayed by HPLC [18]) at a final concentration of 26 lM was used to evaluate the activity of the system towards MSSM. The activity of the thioredoxin system was measured by spectrophotometry in two procedures.…”

“…All the biochemical assays were performed in triplicate. Formation of products from each substrate was analyzed by HPLC according to published protocols [18]. Briefly, reactions were performed at 30°C, and aliquots of 200 ll were taken at time 0, 10, and 60 min.…”

“…HPLC analysis was performed [18] to directly measure the amount of product formed in the enzymatic reaction, and to comparatively assess the oxidoreductase activity of the thioredoxin system towards GSSG, GSNO and MSSM. In the presence of TrxC, TrxR, and NADPH, GSH was produced from both GSSG and GSNO ( Fig.…”