Fig. 2: Binding of AcuA to AcsA inactivates AcsA activity and acetylation of AcsA at K549 by AcuA results in dissociation of AcuA from AcsA. | Nature Communications

Fig. 2: Binding of AcuA to AcsA inactivates AcsA activity and acetylation of AcsA at K549 by AcuA results in dissociation of AcuA from AcsA.

From: Acetyl-CoA synthetase activity is enzymatically regulated by lysine acetylation using acetyl-CoA or acetyl-phosphate as donor molecule

Fig. 2

a Binding of AcuA to AcsA inactivates AcsA-activity. The preformed AcsA•AcuA complex (pre) or AcsA alone (post) was incubated with/without CoA (C)/acetyl-CoA (aC), ATP/AMP and acetate, as indicated. Afterwards, AcuA was also added to the post samples and all samples were incubated. The acetyl-CoA generated by acetyl-CoA synthetase (AcsA) activity was detected indirectly by immunoblotting with an anti-acetyl-lysine antibody (IB: AcK) assessing AcsA K549-acetylation. The result was confirmed in at least two independent experiments. Lane M represents the protein molecular weight marker. Source data are provided as Source Data file. b The AcsA•AcuA complex dissociates upon acetylation of AcsA by AcuA in the presence of acetyl-CoA. SEC runs were performed with the AcsA•AcuA complex pretreated with acetyl-CoA. AcsA elutes as homodimer (13.63 ml), AcuA as monomer (17.48 ml). The peak at 19.89 ml corresponds to CoA/acetyl-CoA. Immunoblotting (IB: AcK) shows AcsA lysine acetylation and Ponceau S-red staining (PoS) shows AcsA•AcuA dissociation. Lane + indicates the acetylated B. subtilis AcsA used as technical control, the M represents the protein molecular weight marker. The experiment was repeated independently three times with similar results. Source data are provided as Source Data file. c Acetylation of AcsA K549 is performed by AcuA and can be reversed by AcuC. AcsA or AcsA K549R (10 µM) was incubated with AcuA (2 µM) in the presence/absence of acetyl-CoA (0.5 mM) or the deacetylase AcuC (20 µM), as indicated. Samples lacking AcuC contained deacetylase inhibitor SAHA (50 µM). 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. d AcsA K549Q alters the conformation of the AcsA•AcuA complex. AlphaFold2 structure predictions suggest conformational changes in the AcsA•AcuA complex upon mutation of AcsA K549Q. The AcsA K549Q C-terminal domain moves towards AcuA. Q549 is not oriented towards the AcuA active site.

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