title: Characterization of Mycobacterium tuberculosis mycothiol S-conjugate amidase.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093, USA.
authors.forename: Micah
authors.initials: M
authors.lastname: Steffek
authors.forename: Gerald L
authors.initials: GL
authors.lastname: Newton
authors.forename: Yossef
authors.initials: Y
authors.lastname: Av-Gay
authors.forename: Robert C
authors.initials: RC
authors.lastname: Fahey
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D004187:Disaccharides / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D006020:Glycopeptides; D006868:Hydrolysis; D007294:Inositol; D007700:Kinetics; D019216:Metals, Heavy / Q000737:chemistry; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011720:Pyrazoles / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D012294:Rifamycins / Q000096:biosynthesis; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D004187:Disaccharides; D004791:Enzyme Inhibitors; D006020:Glycopeptides; D019216:Metals, Heavy; D011720:Pyrazoles; D011994:Recombinant Proteins; D012294:Rifamycins; D013438:Sulfhydryl Compounds; C089265:mycothiol; D007294:Inositol; D000581:Amidohydrolases; C413523:mycothiol S-conjugate amidase; D003545:Cysteine; C032779:rifamycin S