Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl CoA from acetate, adenosine triphosphate, and CoA through an acetyl-adenosine monophosphate (AMP) intermediate. Immunoblotting and mass spectrometry analysis showed that Salmonella enterica Acs enzyme activity is posttranslationally regulated by acetylation of lysine-609. Acetylation blocks synthesis of the adenylate intermediate but does not affect the thioester-forming activity of the enzyme. Activation of the acetylated enzyme requires the nicotinamide adenine dinucleotide-dependent protein deacetylase activity of the CobB Sir2 protein from S. enterica. We propose that acetylation modulates the activity of all the AMP-forming family of enzymes, including nonribosomal peptide synthetases, luciferase, and aryl- and acyl-CoA synthetases. These findings extend our knowledge of the roles of Sir2 proteins in gene silencing, chromosome stability, and cell aging and imply that lysine acetylation is a common regulatory mechanism in eukaryotes and prokaryotes.

The yeast Sir2 protein, required for transcriptional silencing, has an NAD ؉ -dependent histone deacetylase (HDA) activity. Yeast extracts contain a NAD ؉ -dependent HDA activity that is eliminated in a yeast strain from which SIR2 and its four homologs have been deleted. This HDA activity is also displayed by purified yeast Sir2p and homologous Archaeal, eubacterial, and human proteins, and depends completely on NAD ؉ in all species tested. The yeast NPT1 gene, encoding an important NAD ؉ synthesis enzyme, is required for rDNA and telomeric silencing and contributes to silencing of the HM loci. Null mutants in this gene have significantly reduced intracellular NAD ؉ concentrations and have phenotypes similar to sir2 null mutants. Surprisingly, yeast from which all five SIR2 homologs have been deleted have relatively normal bulk histone acetylation levels. The evolutionary conservation of this regulated activity suggests that the Sir2 protein family represents a set of effector proteins in an evolutionarily conserved signal transduction pathway that monitors cellular energy and redox states. T ranscriptional silencing is a regulatory mechanism that results in the inactivation of large blocks of chromosomes via an altered chromatin structure. In Saccharomyces cerevisiae, silencing is observed at the HM silent mating type loci (reviewed in ref. 1), telomeres (2), and at the rDNA locus (3, 4). Although a different subset of proteins is required for silencing at each of the three loci, all types of silencing require Sir2p (3, 5). The Sir2 family of proteins is highly conserved and found in Archaea, eubacteria, and metazoa (6-9). A recent study showed that yeast and mouse Sir2p have NAD ϩ -dependent HDA activity on histone peptides specific for Lys-16 of histone H4 (10), an important residue for silencing (11-13). Earlier work had suggested that Sir2p might have HDA activity. Acetylated histones were inefficiently immunoprecipitated from the silent mating type (HM) loci relative to the expressed mating type (MAT) locus, and overexpression of Sir2p led to changes in levels of bulk histone acetylation (14,15). Other recent papers demonstrated a phosphotransferase activity for Sir2p, with NAD ϩ as the source of phosphate and a variety of proteins implicated as targets of ADP ribosylation (9, 16). A sir2 missense mutation that destroys this in vitro activity also destroys silencing in vivo. These results suggest that the Sir2p family is a group of ADP-ribosyl transferases (ARTs).We show here that Archaeal, eubacterial, and human Sir2 proteins, like Sir2p, have potent NAD ϩ -dependent HDA activity in vitro. The importance of NAD ϩ to the in vivo activity of Sir2p is underscored by our finding that mutations in the S. cerevisiae NPT1 gene lead to severe silencing defects. NPT1 encodes a nicotinate phosphoribosyltransferase, required for NAD ϩ synthesis through a salvage pathway. Intracellular NAD ϩ levels are significantly lower in npt1 null mutants than in the wild type, providing independent evidence that NAD ϩ is critic...

Acetyl-coenzyme A synthetase catalyzes the two-step synthesis of acetyl-CoA from acetate, ATP, and CoA and belongs to a family of adenylate-forming enzymes that generate an acyl-AMP intermediate. This family includes other acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular nonribosomal peptide synthetases. We have determined the X-ray crystal structure of acetyl-CoA synthetase complexed with adenosine-5'-propylphosphate and CoA. The structure identifies the CoA binding pocket as well as a new conformation for members of this enzyme family in which the approximately 110-residue C-terminal domain exhibits a large rotation compared to structures of peptide synthetase adenylation domains. This domain movement presents a new set of residues to the active site and removes a conserved lysine residue that was previously shown to be important for catalysis of the adenylation half-reaction. Comparison of our structure with kinetic and structural data of closely related enzymes suggests that the members of the adenylate-forming family of enzymes may adopt two different orientations to catalyze the two half-reactions. Additionally, we provide a structural explanation for the recently shown control of enzyme activity by acetylation of an active site lysine.