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

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 nonreplicat-ing 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 H 2O 2 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, bioinformat-ics, 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