Reversible protein acetylation is a ubiquitous means for the rapid control of diverse cellular processes. Acetyltransferase enzymes transfer the acetyl group from acetyl-CoA to lysine residues, while deacetylase enzymes catalyze removal of the acetyl group by hydrolysis or by an NAD ؉ -dependent reaction. Propionyl-coenzyme A (CoA), like acetyl-CoA, is a high energy product of fatty acid metabolism and is produced through a similar chemical reaction. Because acetyl-CoA is the donor molecule for protein acetylation, we investigated whether proteins can be propionylated in vivo, using propionyl-CoA as the donor molecule. We report that the Salmonella enterica propionyl-CoA synthetase enzyme PrpE is propionylated in vivo at lysine 592; propionylation inactivates PrpE. The propionyl-lysine modification is introduced by bacterial Gcn-5-related N-acetyltransferase enzymes and can be removed by bacterial and human Sir2 enzymes (sirtuins). Like the sirtuin deacetylation reaction, sirtuin-catalyzed depropionylation is NAD ؉ -dependent and produces a byproduct, O-propionyl ADP-ribose, analogous to the O-acetyl ADP-ribose sirtuin product of deacetylation. Only a subset of the human sirtuins with deacetylase activity could also depropionylate substrate. The regulation of cellular propionylCoA by propionylation of PrpE parallels regulation of acetylCoA by acetylation of acetyl-CoA synthetase and raises the possibility that propionylation may serve as a regulatory modification in higher organisms.Protein acetylation is a ubiquitous means for the rapid control of diverse cellular processes (1, 2). Acetylation occurs at lysine residues, with acetyl-CoA (Ac-CoA) 5 serving as the acetyl group donor. In higher organisms, aberrant acetylation of lysine residues in histone tails correlates with diseases such as cancers and developmental disorders and may contribute to modulation of cell life span (3, 4). From bacteria to humans, N-Lys acetylation controls the activity of acetyl-coenzyme A synthetase (AMP-forming; Acs) and potentially that of other members of the AMP-forming family of enzymes (5-7). In Salmonella enterica, Acs is deacetylated by CobB, a member of the Sir2 family of NAD ϩ -dependent deacetylases (a.k.a. sirtuins) (8). Interestingly, strains of S. enterica lacking CobB deacetylase activity cannot grow on propionate because the propionyl-CoA synthetase (encoded by the prpE gene) that activates propionate to propionyl-CoA is not active (5, 9).Cells generate propionyl-CoA from several different processes, including the catabolism of odd chain fatty acids, the decarboxylation of succinate, the catabolism of amino acids (e.g. threonine), and the activation of propionate (10 -12). Propionate is a powerful inhibitor of cell growth that is widely used as a food preservative. Reports in the literature suggest that propionyl-CoA may be responsible for the cytotoxic effects of propionate. Although the direct effects of propionyl-CoA are unclear, it is clear that cells avoid accumulating this metabolite (13-15). The cell maintains...

SummaryAcetyl-coenzyme A synthetase (Acs) activates acetate into acetyl-coenzyme A (Ac-CoA) in most cells. In Salmonella enterica, acs expression and Acs activity are controlled. It is unclear why the sirtuin-dependent protein acylation/deacylation system (SDPADS) controls the activity of Acs. Here we show that, during growth on 10 mM acetate, acs + induction in a S. enterica strain that cannot acetylate (i.e. inactivate) Acs leads to growth arrest, a condition that correlates with a drop in energy charge (0.17) in the acetylationdeficient strain, relative to the energy charge in the acetylation-proficient strain (0.71). Growth arrest was caused by elevated Acs activity, a conclusion supported by the isolation of a single-amino-acid variant (Acs G266S ), whose overproduction did not arrest growth. Acs-dependent depletion of ATP, coupled with the rise in AMP levels, prevented the synthesis of ADP needed to replenish the pool of ATP. Consistent with this idea, overproduction of ADP-forming Ac-CoAsynthesizing systems did not affect the growth behaviour of acetylation-deficient or acetylation-proficient strains. The Acs G266S variant was > 2 orders of magnitude less efficient than the Acs WT enzyme, but still supported growth on 10 mM acetate. This work provides the first evidence that SDPADS function helps cells maintain energy homeostasis during growth on acetate.

This report shows that Salmonella enterica catabolizes ethanolamine to acetyl-CoA (Ac-CoA), which enters the glyoxylate bypass and tricarboxylic acid cycle for the generation of energy and central metabolites. During growth on ethanolamine, S. enterica excreted acetate, whose recapture depended on Ac-CoA synthetase (Acs) and the housekeeping phosphotransacetylase (Pta) enzyme activities. The Pta enzyme did not play a role in acetate excretion during growth of S. enterica on ethanolamine. It is proposed that during growth on ethanolamine, acetate excretion is necessary to maintain a pool of free CoA. Acetate excretion requires the eut operon-encoded phosphotransacetylase (EutD) and acetate kinase (Ack) enzymes. EutD function was not required for growth on ethanolamine, and an eutD strain showed only a slight reduction in growth rate. The existence of an as-yet-unidentified system that releases acetate was revealed during growth of a strain lacking Acs, the housekeeping phosphotransacetylase (Pta), and EutD. The functions of pyruvate oxidase (PoxB), Ack and STM3118 protein [a homologue of the Saccharomyces cerevisiae Ac-CoA hydrolase (Ach1p) enzyme] were not involved in the release of acetate by the acs pta eutD strain.