mycothiol and plumbagin

Here we show that a series of inhibitors, constructed from plumbagin conjugated to a phenyl thioglucoside via an alkyl chain of variable length, are bound in solution-favoured ligand conformations to a mycothiol biosynthetic enzyme MshB, a GlcNAc-Ins deacetylase. The kinetic studies of this ligand series show that MshB is more strongly inhibited as a function of increasing alkyl chain length. While docking studies yielded highest ranked conformations in which the ligands extended along the catalytic site, these conformations produced free energy values prone to large errors and which were inconsistent with experimental kinetic measurements. Solution-favoured conformations of the inhibitors feature a preference for intramolecular aromatic association that results in curled conformations. Free energy perturbation calculations of MshB bound to the inhibitors in the preconfigured solution-favoured curled conformations gave the same binding pattern observed in the kinetic experiments. On investigation of these conformations lodged in the catalytic domain, we found that the selective feature determining their relative binding strength was the result of an optimisation of the dispersion interactions between the ligand aromatic groups phenyl and plumbagin, and the enzyme aromatic groups His144 and Tyr142 respectively. These results show that rather than deform the preferred folded ligand solution conformation, such that the hydrophobic C-2 acyl chain is linearly projected into a buried hydrophobic rich binding cavity adjacent to the active site, MshB binds preconfigured solution inhibitor curled conformations with a preference for aromatic association.

Topics: Amidohydrolases; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Cysteine; Enzyme Inhibitors; Glycopeptides; Inositol; Kinetics; Ligands; Molecular Conformation; Molecular Docking Simulation; Mycobacterium tuberculosis; Naphthoquinones; Protein Structure, Secondary; Superoxides; Thermodynamics; Thioglucosides

N-Acetylglucosaminylinositol (GlcNAc-Ins)-deacetylase (MshB) and mycothiol-S-conjugate amidase (Mca), structurally related amidases present in mycobacteria and other Actinomycetes, are involved in the biosynthesis of mycothiol and in the detoxification of xenobiotics as their mycothiol-S-conjugates, respectively. With substrate analogs of GlcNAc-Ins, MshB showed a marked preference for inositol as the aglycon present in GlcNAc-Ins. The inhibition of MshB and Mca by 10 thioglycosides, 7 cyclohexyl-2-deoxy-2-C-alkylglucosides, and 4 redox cyclers was evaluated. The latter contained plumbagin tethered via 2 to 5 methylene carbons and an amide linkage to phenyl-2-deoxy-2-amino-1-thio-alpha-d-glucopyranoside. These proved to be the most potent amongst the 21 compounds tested as inhibitors of MshB. Their inhibitory potency varied with the length of the spacer, with the compound with longest spacer being the most effective.

Topics: Acetylcysteine; Amidohydrolases; Bacterial Proteins; Cell Survival; Cysteine; Enzyme Inhibitors; Glycopeptides; Indicators and Reagents; Inositol; Mycobacterium tuberculosis; NADH, NADPH Oxidoreductases; Naphthoquinones; Oxidation-Reduction; Structure-Activity Relationship; Substrate Specificity; Thioglucosides

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.

Topics: Amidohydrolases; Anti-Bacterial Agents; Bridged Bicyclo Compounds; Cysteine; Dinitrochlorobenzene; Disaccharides; Enzyme Inhibitors; Ethylmaleimide; Gene Deletion; Genes, Bacterial; Genetic Complementation Test; Glycopeptides; Inositol; Iodoacetamide; Mutagenesis, Insertional; Mycobacterium smegmatis; Mycobacterium tuberculosis; Naphthoquinones; Oxidants; Pyrazoles; Rifamycins; Streptomycin; Sulfhydryl Compounds; Vitamin K 3