Summary GCN 5‐type N ‐acetyltransferases ( GNATs ) are enzymes that catalyse the transfer of the acetyl group from acetyl‐ CoA to a primary amine. GNATs are conserved in all domains of life. Some members of this family of enzymes acetylate the side‐chain of specific lysine residues in proteins of diverse function. In bacteria, GNAT ‐catalysed protein acetylation regulates carbon metabolism, RNA metabolism and transcriptional regulation. Metabolic regulation in S treptomyces species is of interest due to the role of these organisms in natural product synthesis. Here we identify Sl PatA , a GNAT in S treptomyces lividans with unique domain organization, and a new acetylation target, namely acetoacetyl‐ CoA synthetase ( Sl AacS ). The latter has homologues in all domains of life. In vitro and in vivo evidence show that Sl AacS is a bona fide acetoacetyl‐ CoA synthetase. Sl PatA acetylates Sl AacS more efficiently than it does acetyl‐ CoA synthetase, an enzyme known to be under acetylation control. Sl PatA acetylates Sl AacS at the active‐site residue Lys 617 and acetylation inactivates Sl AacS . Acetylated Sl AacS was deacetylated by a sirtuin‐type protein deacetylase. Sl AacS acetylation/deacetylation may represent a conserved mechanism for regulation of acetoacetyl‐ CoA synthetase activity in all domains of life.