{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Tsuji K"],"funding":["JSPS KAKENHI","World Premier International Research Centre Initiative"],"pagination":["53-73"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC6325607"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["9(1)"],"pubmed_abstract":["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 H<sub>2</sub>O<sub>2</sub> and NaOCl, which resulted in irreversible enzyme inactivation. By contrast, treatment with coenzyme A disulphide (CoASSCoA) or H<sub>2</sub>O<sub>2</sub>/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."],"journal":["FEBS open bio"],"pubmed_title":["Glyceraldehyde-3-phosphate dehydrogenase from <i>Citrobacter</i> sp. S-77 is post-translationally modified by CoA (protein CoAlation) under oxidative stress."],"pmcid":["PMC6325607"],"funding_grant_id":["JP18H02091","JP26000008"],"pubmed_authors":["Yoon KS","Ogo S","Tsuji K"],"additional_accession":[]},"is_claimable":false,"name":"Glyceraldehyde-3-phosphate dehydrogenase from <i>Citrobacter</i> sp. S-77 is post-translationally modified by CoA (protein CoAlation) under oxidative stress.","description":"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 H<sub>2</sub>O<sub>2</sub> and NaOCl, which resulted in irreversible enzyme inactivation. By contrast, treatment with coenzyme A disulphide (CoASSCoA) or H<sub>2</sub>O<sub>2</sub>/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.","dates":{"release":"2019-01-01T00:00:00Z","publication":"2019 Jan","modification":"2026-05-07T14:43:00.865Z","creation":"2019-03-26T22:37:41Z"},"accession":"S-EPMC6325607","cross_references":{"pubmed":["30652074"],"doi":["10.1002/2211-5463.12542"]}}