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-gluc opyranoside (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 smegmati s was treated with the alkylating agent monobromobimane (mBBr), the cellula r mycothiol was converted to its bimane derivative (MSmB). The latter was r apidly cleaved to produce GlcN-Ins and the bimane derivative of N-acetylcys teine (AcCySmB), a mercapturic acid that was rapidly exported from the cell s 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-con jugate amidase (amidase) responsible for cleaving MSmB was purified to homo geneity from M. smegmatis. A value of K-m = 95 +/- 8 mu M and a value of k( cat) = 8 s(-1) was determined for the amidase with MSmB as substrate. Activ ity with 100 mu M mycothiol or with the monobromobimane derivative of 1-D-m yo-inosityl-2-(L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (CySmB-Glc N-Ins) or of 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-(alpha,beta)-D-glucopyra noside (AcCySmB-GlcN) was at least 10(3) lower than with 100 mu M MSmB, dem onstrating that the amidase is highly specific for S-conjugates of mycothio l. Conjugates of mycothiol with the antibiotic cerulenin, N-ethylmaleimide, 3-(N-maleimidopropionyl)-biocytin, and 7-diethylamino-3-(4'-maleimidylphen yl)-4-methylcoumarin also exhibited significant activity, The sequence of t he amino-terminal 20 residues was determined, and an open reading frame (Rv 1082) 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 Es cherichia coli, and the expressed protein was shown to have substrate speci ficity similar to the amidase from M. smegmatis. These results indicate tha t mycothiol and mycothiol S-conjugate amidase play an important role in the detoxification of alkylating agents and antibiotics.