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|Title:||The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis||Authors:||Rosado, Leonardo Astolfi
de la Rubia, Alfonso G
Guerin, Marcelo E
Mateos, Luis M
|Keywords:||Mycobacterium tuberculosis;enzyme catalysis;oxidation-reduction (redox);oxidative stress;oxidoreductase;phylogenetics;thiol–disulfide exchange||Keywords Plus:||ESCHERICHIA-COLI THIOREDOXIN;METHIONINE SULFOXIDE REDUCTASE;CORYNEBACTERIUM-GLUTAMICUM;THIOL REDOX;MAXIMUM-ENTROPY;GLUTAREDOXIN;PROTEIN;DISULFIDE;MECHANISM;RESISTANCE||Mesh headings:||Anti-Bacterial Agents;Bacterial Proteins;Models, Molecular;Mycobacterium tuberculosis;Oxidative Stress;Prodrugs;Protein Disulfide-Isomerases;Pyrimidines||Secondary Mesh headings:||Activation, Metabolic;Biocatalysis;Catalytic Domain;Crystallography, X-Ray;Cysteine;Disk Diffusion Antimicrobial Tests;Drugs, Investigational;Gene Deletion;Molecular Conformation;Molecular Docking Simulation;Oxidation-Reduction;Phylogeny;Protein Conformation;Recombinant Proteins;Substrate Specificity||Issue Date:||2017||Publisher:||AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC||Journal:||The Journal of biological chemistry||Abstract:||
The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S-mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.
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