• Acetyl phosphate (AcP) directly acetylates and phosphorylates E. coli pyruvate dehydrogenase (AceE), modulating its enzymatic activity.
• CobB deacetylase reverses AcP-mediated acetylation of AceE, highlighting a reversible regulatory mechanism.
• Deletion of ackA or pta alters AceE acetylation and activity, leading to pyruvate accumulation and changes in pyruvate metabolism gene expression.
• This study establishes a novel link between AcP-mediated protein acetylation, pyruvate dehydrogenase activity, and pyruvate metabolism in E. coli.