Pausing protein acetylation by an Ac-CoA synthetase/acetyltransferase complex.

Acetyl-CoA synthetase (Acs) generates acetyl-Coenzyme A (Ac-CoA) but its excessive activity can deplete ATP and lead to growth arrest. To prevent this, Acs is tightly regulated through Ac-CoA-dependent feedback inhibition executed by Ac-CoA-dependent acetyltransferases such as AcuA in Bacillus subtilis. AcuA acetylates the catalytic lysine of AcsA turning the synthetase inactive. Here we report an Ac-CoA independent inhibition mechanism of AcsA, which relies on a complex between AcsA and AcuA. Our structural analysis reveals that AcuA and AcsA form a tightly intertwined complex – the C-terminal domain binds to acetyltransferase domain of AcuA, while the C-terminus of AcuA occupies the CoA-binding site in the N-terminal domain of AcsA. Our structure-guided biochemical analysis reveals that AcuA inhibits AcsA by an Ac-CoA-independent inhibition via the AcsA-AcuA complex, in addition to the well-known Ac-CoA-dependent inhibition via acetylation of the catalytic lysine 549 in AcsA disrupting the complex. These findings elucidate how AcuA specifically binds to its target and inhibits activity through an unprecedented Ac-CoA-independent mechanism when Ac-CoA levels are low. Our study suggests that the two mechanistically distinct inhibitory mechanisms accomplished by AcuA adjust AcsA activity to the cellular concentrations of the different substrates of the reaction, illustrating the complexity underlying the regulatory framework of acetyl-CoA synthesis from acetate.