biostudies-literatureTsuji KJSPS KAKENHIWorld Premier International Research Centre Initiative53-73https://www.ebi.ac.uk/biostudies/studies/S-EPMC6325607biostudies-literatureUnknown9(1)Protein CoAlation (S-thiolation by coenzyme A) has recently emerged as an alternative redox-regulated post-translational modification by which protein thiols are covalently modified with coenzyme A (CoA). However, little is known about the role and mechanism of this post-translational modification. In the present study, we investigated CoAlation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a facultative anaerobic Gram-negative bacterium <i>Citrobacter</i> sp. S-77 (<i>Cb</i> GAPDH). GAPDH is a key glycolytic enzyme whose activity relies on the thiol-based redox-regulated post-translational modifications of active-site cysteine. LC-MS/MS analysis revealed that CoAlation of <i>Cb</i> GAPDH occurred <i>in vivo</i> under sodium hypochlorite (NaOCl) stress. The purified <i>Cb</i> GAPDH was highly sensitive to overoxidation by H₂O₂ and NaOCl, which resulted in irreversible enzyme inactivation. By contrast, treatment with coenzyme A disulphide (CoASSCoA) or H₂O₂/NaOCl in the presence of CoA led to CoAlation and inactivation of the enzyme; activity could be recovered after incubation with dithiothreitol, glutathione and CoA. CoAlation of the enzyme <i>in vitro</i> was confirmed by mass spectrometry. The presence of a substrate, glyceraldehyde-3-phosphate, fully protected <i>Cb</i> GAPDH from inactivation by CoAlation, suggesting that the inactivation is due to the formation of mixed disulphides between CoA and the active-site cysteine Cys149. A molecular docking study also supported the formation of mixed disulphide without steric constraints. These observations suggest that CoAlation is an alternative mechanism to protect the redox-sensitive thiol (Cys149) of <i>Cb</i> GAPDH against irreversible oxidation, thereby regulating enzyme activity under oxidative stress.FEBS open bioGlyceraldehyde-3-phosphate dehydrogenase from <i>Citrobacter</i> sp. S-77 is post-translationally modified by CoA (protein CoAlation) under oxidative stress.PMC6325607JP18H02091JP26000008Yoon KSOgo STsuji KfalseGlyceraldehyde-3-phosphate dehydrogenase from <i>Citrobacter</i> sp. S-77 is post-translationally modified by CoA (protein CoAlation) under oxidative stress.Protein CoAlation (S-thiolation by coenzyme A) has recently emerged as an alternative redox-regulated post-translational modification by which protein thiols are covalently modified with coenzyme A (CoA). However, little is known about the role and mechanism of this post-translational modification. In the present study, we investigated CoAlation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a facultative anaerobic Gram-negative bacterium <i>Citrobacter</i> sp. S-77 (<i>Cb</i> GAPDH). GAPDH is a key glycolytic enzyme whose activity relies on the thiol-based redox-regulated post-translational modifications of active-site cysteine. LC-MS/MS analysis revealed that CoAlation of <i>Cb</i> GAPDH occurred <i>in vivo</i> under sodium hypochlorite (NaOCl) stress. The purified <i>Cb</i> GAPDH was highly sensitive to overoxidation by H₂O₂ and NaOCl, which resulted in irreversible enzyme inactivation. By contrast, treatment with coenzyme A disulphide (CoASSCoA) or H₂O₂/NaOCl in the presence of CoA led to CoAlation and inactivation of the enzyme; activity could be recovered after incubation with dithiothreitol, glutathione and CoA. CoAlation of the enzyme <i>in vitro</i> was confirmed by mass spectrometry. The presence of a substrate, glyceraldehyde-3-phosphate, fully protected <i>Cb</i> GAPDH from inactivation by CoAlation, suggesting that the inactivation is due to the formation of mixed disulphides between CoA and the active-site cysteine Cys149. A molecular docking study also supported the formation of mixed disulphide without steric constraints. These observations suggest that CoAlation is an alternative mechanism to protect the redox-sensitive thiol (Cys149) of <i>Cb</i> GAPDH against irreversible oxidation, thereby regulating enzyme activity under oxidative stress.2019-01-01T00:00:00Z2019 Jan2026-05-07T14:43:00.865Z2019-03-26T22:37:41ZS-EPMC63256073065207410.1002/2211-5463.12542