The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of .

TitleThe antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of .
Publication TypeJournal Article
Year of Publication2017
AuthorsRosado, L. Astolfi, K. Wahni, G. Degiacomi, B. Pedre, D. Young, A. G. de la Rubia, F. Boldrin, E. Martens, L. Marcos-Pascual, E. Sancho-Vaello, D. Albesa-Jové, R. Provvedi, C. Martin, V. Makarov, W. Versées, G. Verniest, M. E. Guerin, L. M. Mateos, R. Manganelli, and J. Messens
JournalJ Biol Chem
Volume292
Issue32
Pagination13097-13110
Date Published2017 08 11
ISSN1083-351X
KeywordsActivation, Metabolic, Anti-Bacterial Agents, Bacterial Proteins, Biocatalysis, Catalytic Domain, Crystallography, X-Ray, Cysteine, Disk Diffusion Antimicrobial Tests, Drugs, Investigational, Gene Deletion, Models, Molecular, Molecular Conformation, Molecular Docking Simulation, Mycobacterium tuberculosis, Oxidation-Reduction, Oxidative Stress, Phylogeny, Prodrugs, Protein Conformation, Protein Disulfide-Isomerases, Pyrimidines, Recombinant Proteins, Substrate Specificity
Abstract

The 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 have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under HO 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 -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 (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.

DOI10.1074/jbc.M117.797837
Alternate JournalJ. Biol. Chem.
PubMed ID28620052
PubMed Central IDPMC5555174
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