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J Biol Chem. 2017 Aug 11;292(32):13097-13110. doi: 10.1074/jbc.M117.797837. Epub 2017 Jun 15.

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

Author information

1
From the Center for Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), B-1050 Brussels, Belgium.
2
the Brussels Center for Redox Biology, B-1050 Brussels, Belgium.
3
Structural Biology Brussels and.
4
the Departments of Molecular Medicine and.
5
the Department of Molecular Biology, Area of Microbiology, University of León, 24071 León, Spain.
6
the Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC, UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain.
7
the Departamento de Bioquímica, Universidad del País Vasco, Leioa, Bizkaia 48940, Spain.
8
the Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain.
9
IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain, and.
10
Biology, University of Padova, 35121 Padova, Italy.
11
the Research Group of Organic Chemistry, Vrije Universiteit Brussel, B-1050 Brussels, Belgium.
12
the A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia.
13
the Departments of Molecular Medicine and riccardo.manganelli@unipd.it.
14
From the Center for Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), B-1050 Brussels, Belgium, joris.messens@vib-vub.be.

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.

KEYWORDS:

Mycobacterium tuberculosis; enzyme catalysis; oxidation-reduction (redox); oxidative stress; oxidoreductase; phylogenetics; thiol–disulfide exchange

PMID:
28620052
PMCID:
PMC5555174
DOI:
10.1074/jbc.M117.797837
[Indexed for MEDLINE]
Free PMC Article

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