The glyceraldehyde-3-phosphate dehydrogenase GapDH of Corynebacterium diphtheriae is redox-controlled by protein S-mycothiolation under oxidative stress

Abstract

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification 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 modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 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.

Glyceraldehyde‐3‐phosphate dehydrogenase from Citrobacter sp. S‐77 is post‐translationally modified by CoA (protein CoAlation) under oxidative stress

“…The structural determinant for the preference of these redox modifications (S-thiolation or intramolecular disulphide) remains to be elucidated. Cys149 is known as a conserved cysteine for substrate binding and as the modification site of sulphenation and S-thiolation of GAPDHs among all domains of life [27,28,33,34,45]. In fact, the extreme vulnerability of Cys149 in CbGAPDH to H 2 O 2 was observed, which is consistent with the findings of previous studies [27,28,33,34,45].…”

“…Typically, oxidation of the active‐site cysteine (Cys149) of GAPDH yields sulphenate and subsequently yields sulphinate ( ) or sulfonate ( ) by overoxidation . In contrast to the latter two forms, sulphenate is a transient intermediate towards the formation of intramolecular disulphide or mixed disulphides with cellular low‐molecular‐weight thiols such as GSH, which results in reversible inactivation to prevent irreversible oxidation of GAPDH . We thus examined the effects of H 2 O 2 with/without CoA on Cb GAPDH activity to investigate whether CoA can protect Cb GAPDH against irreversible oxidation by CoAlation.…”

“…The intramolecular disulphide bonding (Cys149‐S‐S‐Cys153) was also detected as an alternative form of redox regulation of the enzyme. The formation of intramolecular disulphide bonding has been reported in several studies . The structural determinant for the preference of these redox modifications (S‐thiolation or intramolecular disulphide) remains to be elucidated.…”

“…Besides the CoASSCoA-dependent mechanism, initial oxidation of labile cysteine to sulphenate followed by reaction with low-molecular-weight thiol is proposed as a plausible mechanism of S-thiolation [25,27,[32][33][34]45]. Indeed, CbGAPDH The formation of intramolecular disulphide bonding has been reported in several studies [28,30,32,47]. The structural determinant for the preference of these redox modifications (S-thiolation or intramolecular disulphide) remains to be elucidated.…”

“…Although the mechanism of S-thiolation in vivo remains controversial, GAPDH is known to undergo S-glutathionylation nonenzymatically by incubation with glutathione disulphide (GSSG), H 2 O 2 plus GSH, or nitrosoglutathione (GSNO) in vitro, which results in reversible enzyme inactivation due to modification of catalytic cysteine [32][33][34][35]. S-thiolated GAPDH homologs are enzymatically reactivated by glutaredoxin (Grx), thioredoxin (Trx) or its analogous systems, which are highly conserved across all living organisms [27,28,32,33,[36][37][38]. The consequence of S-thiolation of GAPDH is inhibition of its glycolytic activity and downregulation of glycolysis, which results in redirection to the pentose phosphate pathway for providing a reducing equivalent of NAPDH as a means of adaptation under oxidative stress [29,39].…”