1 Scientific RepoRts | 7: 5020 | DOI:10.1038/s41598-017-05206-2 www.nature.com/scientificreports The glyceraldehyde-3-phosphate dehydrogenase GapDH of Corynebacterium diphtheriae is redox-controlled by protein S- mycothiolation under oxidative stress Melanie Hillion 1 , Marcel Imber 1 , Brandán pedre 2,3,4 , Jörg Bernhardt 5 , Malek saleh 1 , Vu Van Loi 1 , Sandra Maaß 5 , Dörte Becher 5 , Leonardo Astolf Rosado 2,3,4 , Lorenz Adrian 6 , Christoph Weise 7 , Rüdiger Hell 8 , Markus Wirtz 8 , Joris Messens 2,3,4 & Haike Antelmann 1 Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modifcation by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modifed at its active site Cys153 in vivo. Exposure of purifed GapDH to H 2 o 2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H 2 o 2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways. Bacteria are exposed to various redox-active compounds, such as reactive oxygen species (ROS) in their nat- ural habitat or during infections and are equipped with specifc protection mechanisms 1 . To cope with ROS, bacteria use different antioxidant enzymes, such as catalases, peroxiredoxins, superoxide dismutase and low molecular weight (LMW) thiols to maintain the reduced state of the cytoplasm and to survive oxidative stress 2–4 . Gram-negative bacteria utilize glutathione (GSH) as their major LMW thiol, but GSH is absent in most Gram-positive bacteria. Instead, the Actinomycetes that include streptomycetes, corynebacteria and myco- bacteria produce mycothiol (MSH) as their major LMW thiol 5 . MSH functions in detoxifcation of various redox-active compounds, including ROS, electrophiles and antibiotics in all Actinomycetes 6–8 . Apart from its 1 Institute for Biology-Microbiology, Freie Universität Berlin, D-14195, Berlin, Germany. 2 Center for Structural Biology, VIB, B-1050, Brussels, Belgium. 3 Brussels Center for Redox Biology, B-1050, Brussels, Belgium. 4 Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium. 5 institute for Microbiology, ernst-Moritz- Arndt-University of Greifswald, D-17487, Greifswald, Germany. 6 Department Isotope Biogeochemistry, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany. 7 Institute for Chemistry and Biochemistry, Freie Universität Berlin, D-14195, Berlin, Germany. 8 Plant Molecular Biology, Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany. Melanie Hillion, Marcel Imber, Brandán Pedre and Jörg Bernhardt contributed equally to this work. Correspondence and requests for materials should be addressed to H.A. (email: haike.antelmann@fu-berlin.de) Received: 28 March 2017 Accepted: 1 June 2017 Published: xx xx xxxx OPEN