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Structural Basis of Cysteine Ligase MshC Inhibition by Cysteinyl-Sulfonamides.

ABSTRACT: Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC50 values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.

SUBMITTER: Pang L

PROVIDER: S-EPMC9736012 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Structural Basis of Cysteine Ligase MshC Inhibition by Cysteinyl-Sulfonamides.

Pang Luping L Lenders Stijn S Osipov Evgenii M EM Weeks Stephen D SD Rozenski Jef J Piller Tatiana T Cappoen Davie D Strelkov Sergei V SV Van Aerschot Arthur A

International journal of molecular sciences 20221201 23

Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were sy ...[more]

PMID: 36499418

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Project description:Mycobacterium tuberculosis and other members of the actinomycete family produce mycothiol (MSH or acetylcysteine-glucosamine-inositol, AcCys-GlcN-Ins) to protect the organism against oxidative and antibiotic stress. The biosynthesis of MSH proceeds via a five-step process that involves four unique enzymes, MshA-D, which represent specific targets for inhibitor design. Recombinant Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of glucosamine-inositol (GlcN-Ins) and cysteine to form Cys-GlcN-Ins. The 1.6 A three-dimensional structure of MshC in complex with a tight binding bisubstrate analogue, 5'-O-[N-(L-cysteinyl)sulfamonyl]adenosine (CSA), has suggested specific roles for T46, H55, T83, W227, and D251. In addition, a catalytic role for H55 has been proposed on the basis of studies of related aminoacyl-tRNA synthetases. Site-directed mutagenesis was conducted to evaluate the functional roles of these highly conserved residues. All mutants exhibited significantly decreased k(cat) values, with the exception of T83V for which a <7-fold decrease was observed compared to that of the wild type (WT). For the T46V, H55A, W227F, and D251N mutants, the rate of cysteine activation decreased 100-1400-fold compared to that of WT, consistent with the important roles of these residues in the first half-reaction. The approximately 2000-fold decrease in k(cat)/K(m) as well as the approximately 20-fold decrease in K(m) for cysteine suggested a significant role for T46 in cysteine binding. Kinetic studies also indicate a function for W227 in cysteine binding but not in substrate discrimination against serine. H55 was also observed to play a significant role in ATP binding as well as cysteine adenylation. The activity of H55A was partially rescued with exogenous imidazole at acidic pH values, suggesting that the protonated form of histidine is exerting a catalytic role. The pH dependence of the kinetic parameters with the WT enzyme suggests an additional requirement for a catalytic base in cysteinyl ligation.

Dataset Information

Structural Basis of Cysteine Ligase MshC Inhibition by Cysteinyl-Sulfonamides.

Publications

Structural Basis of Cysteine Ligase MshC Inhibition by Cysteinyl-Sulfonamides.

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