Fig. 4: Mutational analyses to confirm the catalytic acetyltransferase mechanism.

a Mutation of the totally conserved residues E102 and W104 of AcuA abolish AcuA AcT activity. AcuA (5 µM) was incubated with acetyl-CoA (200 µM) and AcsA (20 µM) for 3 min, 10 min at 20 °C and 3 h at 37 °C, as indicated. E102A completely abolishes AcT activity of AcuA. W140F shows a reduced AcT activity, while W140A completely abolishes the AcT activity of AcuA towards AcsA. All other mutations do not impair AcT activity. Acetylation of AcsA was assessed by immunoblotting using an anti-AcK-AB (IB: AcK). Loading control (LC) was performed by Ponceau S-red staining (LC: PoS). The lane labelled with M represents the protein molecular weight marker. The result was confirmed in at least three independent experiments. Source data are provided as Source Data file. b Catalytic mechanism proposed for AcT activity of AcuA on K549 of AcsA. MD simulations and mutational analyses suggest that E102 of AcuA acts as a general base/acid during catalysis. W140 is important for acetyl-CoA binding. E102 of AcuA abstracts a proton from K549 of AcsA. This enhances the nucleophilicity of K549 to attack the electrophilic carbon of the acetyl-group of acetyl-CoA. A tetrahedral intermediate is formed and stabilized by the main chain amide of W140 forming an oxyanion hole. The protonated E102 acts as catalytic acid to protonate the CoA thiolate leaving group resulting in the collapse of the intermediate to finally result in acetylated K549 and CoA.