Fig. 6: AcuA acetylation of AcsA K549 in the presence of AcP and CoA results in AcsA•AcuA dissociation. | Nature Communications

Fig. 6: AcuA acetylation of AcsA K549 in the presence of AcP and CoA results in AcsA•AcuA dissociation.

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

Fig. 6

a AcuA (5 µM) acetylates AcsA (25 µM) in the presence of CoA and AcP independent of AcuB (25 µM). Without AcuA no acetylation of AcsA is detectable neither in the presence of AcP (2 mM) alone nor in the presence of AcP and CoA (2 mM). As a control, acetylation of AcsA is observed in the presence of acetyl-CoA (1 mM). All samples were incubated for 2 h at 37 °C. AcsA acetylation was detected by immunoblotting (IB: AcK). Lane M: molecular weight marker. Ponceau S-red staining: loading control (LC: PoS). The result was confirmed in at least three independent experiments. Source data are provided as Source Data file. b Acetylation of AcsA by AcuA in the presence of AcP and CoA leads to AcsA•AcuA dissociation. Treatment of AcsA•AcuA with CoA and AcP results in acetylation of AcsA and dissociation of AcsA•AcuA (elution volume: from 13.50 ml to 17.41 ml (AcsA)) as shown by Ponceau S-red staining (PoS). Immunoblotting (IB: AcK) of SEC elution fractions shows acetylation of AcsA. Lane M: molecular weight marker. The experiment was repeated independently three times with similar results. Source data are provided as Source Data file. c Desulfo-CoA cannot replace CoA in AcP-dependent acetylation of AcsA catalysed by AcuA. In the phosphotransacetylase (Pta)-assay, AcuA (10 µM) and AcsA (10 µM) were incubated with/without CoA/desulfo-CoA (dsCoA; 1 mM) and/or AcP (1 mM) for 2 h or 5 h at 37 °C. Acetylation of AcsA is only observed in samples of AcsA•AcuA containing CoA and AcP. AcsA cannot produce acetyl-CoA using desulfo-CoA (Acs-assay). AcsA (10 µM) was incubated with acetic acid (0.5 mM), ATP (0.5 mM) and CoA/dsCoA (250 µM) for 20 min at 37 °C. Afterwards, AcuA (2 µM) was added (10 min, 20 °C) to detect the generated acetyl-CoA. Detection was done by immunoblotting (IB: AcK). Ponceau S-red staining: loading control (LC: PoS). Lane M: molecular weight marker. The result was confirmed in at least two independent experiments. Source data are provided as Source Data file.

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