Sir2-Dependent Activation of Acetyl-CoA Synthetase by Deacetylation of Active Lysine

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Science  20 Dec 2002:
Vol. 298, Issue 5602, pp. 2390-2392
DOI: 10.1126/science.1077650


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 thatSalmonella 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.

Members of the Sir2 family of proteins (sirtuins) are nicotinamide adenine dinucleotide (NAD+)–dependent deacetylase enzymes involved in chromosome stability, gene silencing, and cell aging in eukaryotes and archaea (1–3). All previously known substrates of sirtuins are components of chromatin and/or affect gene transcription. Strains of the enterobacterium S. entericalacking sirtuin (encoded by the cobB gene) cannot grow on propionate and in low acetate concentration as carbon and energy sources because the acyl-CoA synthetases responsible for converting free acids into acyl-CoA derivatives are inactive (4,5). Our work shows that acetyl-CoA synthetase activity (Fig. 1A) (encoded by the acs gene) is regulated by posttranslational acetylation. We also show that activation of acetylated Acs requires CobB deacetylase activity.

Figure 1

(A) Synthesis of acetyl-CoA by Acs. R, structure of the acid including Cα. (B) The presence of acetylated residues in inactive Acs was established by immunoblotting with polyclonal antibody to N-acetyl-lysine.cobB + and cobB indicate the genotype of the strain from which Acs was isolated. Acs fromcobB cells was incubated with purified CobB as described in (6) for the periods of time indicated.

Acs enzyme synthesized by a cobB strain ofS. enterica was inactive in crude cell-free extract (5). To address the possibility that Acs activity was posttranslationally regulated, we overexpressed the acs gene of S. enterica in cobB + andcobB strains and purified the protein as described in (6). Acs protein produced by thecobB strain was less active than protein isolated from the cobB + strain by a factor of about 100 (Table 1). Incubation of inactive Acs enzyme with purified CobB and NAD+ resulted in a 480-fold increase in Acs activity; the reaction was NAD+ dependent (Table 1, fig. S1). The increase in Acs activity measured in the presence of CobB and the absence of NAD+ was also observed when bovine serum albumin substituted for CobB; thus, this effect appears to be due to nonspecific stabilization of Acs.

Table 1

NAD+-, CobB-dependent activation of acetyl-CoA synthetase.

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We hypothesized that the CobB-dependent activation of Acs was due to removal of acetyl groups from the inactive form of the enzyme. We took two approaches to determine the chemical nature of the modification and the site of the modification in Acs.

We performed immunoblot analysis to determine whether lysyl residues were acetylated in inactive Acs enzyme. Polyclonal antibody to acetyllysine reacted strongly with Acs enzyme isolated from acobB strain (Fig. 1B); in contrast, the same antibody lacked detectable reactivity with Acs isolated from acobB + strain (Fig. 1B). The decreased reactivity of Acs with the antibody depended on the presence of NAD+and CobB in a time-course experiment, establishing a correlation between the lack of Acs activity and acetylation.

We mapped the acetylation site on inactive Acs enzyme by comparing peptide masses generated from tryptic digests of Acs protein isolated from cobB + andcobB cells (fig. S2). The proteolytic mass fingerprints represented >74% of the amino acid sequence from Acs. The mass fingerprints for both active, deacetylated Acs and inactive, acetylated Acs (AcsAc) were virtually identical in peptide masses and their relative intensities, except for a 733.4 ion (fig. S3). This singly charged ion was qualitatively more abundant in the AcsAc preparation than in the Acs preparation, and it corresponds to the predicted mass of the SGKAcIMR612 peptide (7) with acetylation of residue Lys609. We confirmed acetylation of Lys609 by fragmenting the 367.2 ion (the doubly charged species of the 733.4 ion) in Acs or AcsAc tryptic digests (fig. S4). A search of the nonredundant protein database yielded a single hit with the sequence SGKAcIMR612 fromS. enterica Acs with a 0.01 atomic mass unit error on the peptide mass and an error of <21 parts per million on the peptide fragment masses (Table 2, fig. S5). Fragmentation occurs primarily at the peptide bond and yields a b series of ions with the charge on the NH2-terminal amino acid and a y series of ions with the charge on the COOH-terminal amino acid. All but two of the major fragmentation ions present in the fragmentation mass spectra of the 367.2 ion were readily assigned to the SGKAcIMR612 sequence. Most notably, both the y1 to y5 and the b2 to b4 fragment ions were present. These ions span the acetylated Lys609 residue, unambiguously identifying the site of acetylation. These results show that Acs protein overproduced by the cobB strain contains acetyllysine at residue Lys609.

Table 2

Tandem mass spectrometry analysis of the acetylated peptide of Acs.

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To investigate whether acetylation affected both reactions catalyzed by Acs, we took advantage of the knowledge that Acs can synthesize propionyl-CoA from propionate (8). Propionyl–adenosine monophosphate (AMP) (9, 10) was provided as substrate in a reaction mixture containing CoA and Acs or AcsAc. AcsAc enzyme was as efficient as (specific activity = 107 μmol of product per minute per milligram of protein) if not more efficient than the Acs enzyme (specific activity = 73 μmol of product per minute per milligram of protein) in generating propionyl-CoA from propionyl-AMP and CoA. We detected no product in the absence of CoA. These results show that the thioester-forming activity of AcsAc remains unaffected by acetylation and indicate that acetylated residues in AcsAcdo not affect the thioester-forming activity of the enzyme. Acetylation of the active site Lys609 of Acs has the same effect on Acs activity that substitutions of Lys592 have on propionyl-CoA synthetase activity (10). Lys609 of Acs is an invariant residue of a conserved motif in the family of AMP-forming enzymes (Fig. 2). Lys592 of propionyl-CoA synthetase, Lys529 of luciferase, and Lys517 of gramicidin synthetase 1 (equivalent to Lys609 of Acs) are essential for synthesis of the corresponding AMP reaction intermediate but not for the thioester-forming activity of these enzymes (10–13). We propose that acetylation modulates the activity of all the AMP-forming family of enzymes. How these enzymes are acetylated remains an open question.

Figure 2

Conserved motif containing the acetylation site lysine residue among representative members of the AMP-forming family of proteins. Acs, acetyl-CoA synthetase (gi: 16767525, S. enterica); PrpE, propionyl-CoA synthetase (gi: 14917034, S. enterica); Acs2p, acetyl-CoA synthetase (gi: 6323182,Saccharomyces cerevisiae); GrsA, gramicidin S synthetase I (gi: 3334467, Brevibacillus brevis); CepA, one of three subunits that synthesizes chloroeremomycin (European Molecular Biology Laboratory accession numbers X98690 and S46968; gi: 7522085, Amycolotopsis orientalis); cda PSI, calcium-dependent antibiotic peptide synthetase I (open reading frame SCO3230, Streptomyces coelicolor). We used the motif PX4GK to identify putative substrates of sirtuins. gi, GenInfo Identifier.

We provide evidence for a broadened role of sirtuins in cell physiology that includes intermediary metabolism. Our results suggest a mechanism for linking the physiological state of the eukaryotic cell with the acetylation state of histones, a key factor in chromatin silencing and chromosome stability. Several studies implicate sirtuins in life-span control in yeast and metazoans (14,15). Similarly, manipulation of NAD+biosynthetic mechanisms has been shown to affect life-span (16). A recent study documenting the effect of caloric restriction on yeast mother cell longevity suggested that the increased longevity was causally associated with increased respiration; this life-span extension was sirtuin dependent (17). As the Acs enzyme produces acetyl-CoA, a key metabolite of the Krebs cycle, Acs may represent a target for life-span extension.

Supporting Online Material

Materials and Methods

Figs. S1 to S5


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