Identification of the protein acetyltransferase (Pat) enzyme that acetylates acetyl-CoA synthetase in Salmonella enterica

Post-translational modification of proteins is an efficient way cells use to control the activity of structural proteins, gene expression regulatory proteins, and enzymes. In eukaryotes, the Sir2-dependent system of protein acetylation/deacetylation controls a number of processes that affect cell longevity. Sir2 proteins have NAD⁺-dependent protein deacetylase activity and are found in all forms of life. Although the identity of the acetyltransferases that partner with Sir2 enzymes is known in eukaryotes, the identity of the prokaryotic acetyltransferases is not. We report the identification of the gene of Salmonella enterica serovar Typhimurium LT2 encoding the major protein acetyltransferase (Pat) enzyme that, in concert with the CobB sirtuin of this bacterium, regulates the activity of the central metabolic enzyme acetyl-coenzyme A synthetase (Acs). The Pat enzyme uses acetyl-CoA as substrate to modify residue Lys609 of Acs. The Pat/CobB system of S. enterica should serve as the paradigm to further investigate the contributions of this system to the physiology of prokaryotes.     

In Streptomyces lividans,acetyl‐CoA synthetase activity is controlled by O‐serine and Nɛ‐lysine acetylation

Protein acetylation is a rapid mechanism for control of protein function. Acetyl‐CoA synthetase (AMP‐forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK⁶¹⁰). S608 acetylation rendered SlAcs inactive and non‐acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD⁺‐dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin‐dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O‐Ser and N^?‐Lys acetylation.     

In Salmonella enterica, the sirtuin-dependent protein acylation/deacylation system (SDPADS) maintains energy homeostasis during growth on low concentrations of acetate

Acetyl-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 acetylation-deficient 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-CoA-synthesizing 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.     

Purification and characterization of the acetyl-CoA synthetase from Mycobacterium tuberculosis

Acetyl-CoA (AcCoA) synthetase (Acs) catalyzes the conversion of acetate into AcCoA, which is involved in many catabolic and anabolic pathways. Although this enzyme has been studied for many years in many organisms, the properties of Mycobacterium tuberculosis Acs and the regulation of its activity remain unknown. Here, the putative acs gene of M. tuberculosis H37Rv (Mt-Acs) was expressed as a fusion protein with 6×His-tag on the C-terminus in Escherichia coli. The recombinant Mt-Acs protein was successfully purified and then its enzymatic characteristics were analyzed. The optimal pH and temperature, and the kinetic parameters of Mt-Acs were determined. To investigate whether Mt-Acs is regulated by lysine acetylation as reported for Salmonella enterica Acs, its mutant K617R was also generated. Determination of the enzymatic activity suggests that Lys-617 is critical for its function. We further demonstrated that Mt-Acs underwent auto-acetylation with acetate but not with AcCoA as the acetyl donor, which resulted in the decrease of its activity. CoA, the substrate for AcCoA formation, inhibited the auto-acetylation. Furthermore, the silent information regulator (Sir2) of M. tuberculosis (Mt-Sir2) could catalyze Mt-Acs deacetylation, which resulted in activation of Acs. These results may provide more insights into the physiological roles of Mt-Acs in M. tuberculosis central metabolism.     

Staphylococcus aureus modulates the activity of acetyl‐Coenzyme A synthetase (Acs) by sirtuin‐dependent reversible lysine acetylation

Lysine acylation is a posttranslational modification used by cells of all domains of life to modulate cellular processes in response to metabolic stress. The paradigm for the role of lysine acylation in metabolism is the acetyl‐coenzyme A synthetase (Acs) enzyme. In prokaryotic and eukaryotic cells alike, Acs activity is downregulated by acetylation and reactivated by deacetylation. Proteins belonging to the bacterial GCN5‐related N‐acetyltransferase (bGNAT) superfamily acetylate the epsilon amino group of an active site lysine, inactivating Acs. A deacetylase can remove the acetyl group, thereby restoring activity. Here we show the Acs from Staphylococcus aureus (SaAcs) activates acetate and weakly activates propionate, but does not activate >C3 organic acids or dicarboxylic acids (e.g. butyrate, malonate and succinate). SaAcs activity is regulated by AcuA (SaAcuA); a type‐IV bGNAT. SaAcuA can acetylate or propionylate SaAcs reducing its activity by >90% and 95% respectively. SaAcuA also succinylated SaAcs, with this being the first documented case of a bacterial GNAT capable of succinylation. Inactive SaAcs^Ac was deacetylated (hence reactivated) by the NAD⁺‐dependent (class III) sirtuin protein deacetylase (hereafter SaCobB). In vivo and in vitro evidence show that SaAcuA and SaCobB modulate the level of SaAcs activity in S. aureus.     

Acetylation of the chemotaxis response regulator CheY by acetyl-CoA synthetase purified from Escherichia coli

Acetylation of CheY, the excitatory response regulator of bacterial chemotaxis, by the enzyme acetyl-CoA synthetase (Acs) is involved in Escherichia coli chemotaxis, but its function is obscure. Here, we overproduced Acs from E.coli, purified it in quantities sufficient for biochemical work, and characterized both the enzyme and the CheY acetylation reaction that it catalyzes. Such characterization is essential for revealing the function of CheY acetylation in chemotaxis. The enzyme exhibited characteristics typical of prokaryotic Acs enzymes, and it could use either acetate or AcCoA as an acetyl donor for CheY acetylation. The Acs-catalyzed acetylation of CheY was reversible, an essential property for a regulatory process, and cooperative (Hill coefficient approximately 3). By Western blotting with specific anti-acetyl-lysine antibody we demonstrated that Acs undergoes autoacetylation, that CheY is acetylated to a small extent when isolated, and that the extent is elevated following in vitro acetylation. Exposing the intact protein to matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electro-spray mass spectrometry, we found that, in most cases, purified CheY is a mixture of species having zero to six acetyl groups per molecule, with non-acetylated CheY being the most abundant species. By proteolytic in-gel digestion of non-treated CheY followed by peptide fingerprinting, precursor ion scan, and tandem mass spectrometry, we found that the acetylation sites of CheY are clustered at the C terminus of the protein, with lysine residues 91, 92, 109, 119, 122 and 126 being the main acetylation sites. Following in vitro acetylation, the main change that seemed to occur was an incremental increase in the extent of acetylation of the same lysine residues. Thus, CheY is similar to many eukaryotic proteins involved in signaling, which undergo both phosphorylation and multiple acetylation, and in which the acetylation sites are restricted to a particular region.     

Genomewide screen for negative regulators of sirtuin activity in Saccharomyces cerevisiae reveals 40 loci and links to metabolism

Sirtuins are conserved proteins implicated in myriad key processes including gene control, aging, cell survival, metabolism, and DNA repair. In Saccharomyces cerevisiae, the sirtuin Silent information regulator 2 (Sir2) promotes silent chromatin formation, suppresses recombination between repeats, and inhibits senescence. We performed a genomewide screen for factors that negatively regulate Sir activity at a reporter gene placed immediately outside a silenced region. After linkage analysis, assessment of Sir dependency, and knockout tag verification, 40 loci were identified, including 20 that have not been previously described to regulate Sir. In addition to chromatin-associated factors known to prevent ectopic silencing (Bdf1, SAS-I complex, Rpd3L complex, Ku), we identified the Rtt109 DNA repair-associated histone H3 lysine 56 acetyltransferase as an anti-silencing factor. Our findings indicate that Rtt109 functions independently of its proposed effectors, the Rtt101 cullin, Mms1, and Mms22, and demonstrate unexpected interplay between H3K56 and H4K16 acetylation. The screen also identified subunits of mediator (Soh1, Srb2, and Srb5) and mRNA metabolism factors (Kem1, Ssd1), thus raising the possibility that weak silencing affects some aspect of mRNA structure. Finally, several factors connected to metabolism were identified. These include the PAS-domain metabolic sensor kinase Psk2, the mitochondrial homocysteine detoxification enzyme Lap3, and the Fe-S cluster protein maturase Isa2. We speculate that PAS kinase may integrate metabolic signals to control sirtuin activity.     

Identification of a molecular component of the mitochondrial acetyltransferase programme: a novel role for GCN5L1

SIRT3 (sirtuin 3) modulates respiration via the deacetylation of lysine residues in electron transport chain proteins. Whether mitochondrial protein acetylation is controlled by a counter-regulatory program has remained elusive. In the present study we identify an essential component of this previously undefined mitochondrial acetyltransferase system. We show that GCN5L1 [GCN5 (general control of amino acid synthesis 5)-like 1; also known as Bloc1s1] counters the acetylation and respiratory effects of SIRT3. GCN5L1 is mitochondrial-enriched and displays significant homology with a prokaryotic acetyltransferase. Genetic knockdown of GCN5L1 blunts mitochondrial protein acetylation, and its reconstitution in intact mitochondria restores protein acetylation. GCN5L1 interacts with and promotes acetylation of SIRT3 respiratory chain targets and reverses global SIRT3 effects on mitochondrial protein acetylation, respiration and bioenergetics. The results of the present study identify GCN5L1 as a critical prokaryote-derived component of the mitochondrial acetyltransferase programme.     

N-lysine propionylation controls the activity of propionyl-CoA synthetase

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 propionyl-CoA by propionylation of PrpE parallels regulation of acetyl-CoA by acetylation of acetyl-CoA synthetase and raises the possibility that propionylation may serve as a regulatory modification in higher organisms.     

Co-regulation of acetylation and phosphorylation of CheY, a response regulator in chemotaxis of Escherichia coli

CheY, a response regulator of the chemotaxis system in Escherichia coli, can be activated by either phosphorylation or acetylation to generate clockwise rotation of the flagellar motor. Both covalent modifications are involved in chemotaxis, but the function of the latter remains obscure. To understand why two different modifications apparently activate the same function of CheY, we studied the effect that each modification exerts on the other. The phosphodonors of CheY, the histidine kinase CheA and acetyl phosphate, each strongly inhibited both the autoacetylation of the acetylating enzyme, acetyl-CoA synthetase (Acs), and the acetylation of CheY. CheZ, the enzyme that enhances CheY dephosphorylation, had the opposite effect and enhanced Acs autoacetylation and CheY acetylation. These effects of the phosphodonors and CheZ were not caused by their respective activities. Rather, they were caused by their interactions with Acs and, possibly, with CheY. In addition, the presence of Acs elevated the phosphorylation levels of both CheA and CheY, and acetate repressed this stimulation. These observations suggest that CheY phosphorylation and acetylation are linked and co-regulated. We propose that the physiological role of these mutual effects is at two levels: linking chemotaxis to the metabolic state of the cell, and serving as a tuning mechanism that compensates for cell-to-cell variations in the concentrations of CheA and CheZ.     

Short-chain fatty acid activation by acyl-coenzyme A synthetases requires SIR2 protein function in Salmonella enterica and Saccharomyces cerevisiae

SIR2 proteins have NAD(+)-dependent histone deacetylase activity, but no metabolic role has been assigned to any of these proteins. In Salmonella enterica, SIR2 function was required for activity of the acetyl-CoA synthetase (Acs) enzyme. A greater than two orders of magnitude increase in the specific activity of Acs enzyme synthesized by a sirtuin-deficient strain was measured after treatment with homogeneous S. enterica SIR2 protein. Human SIR2A and yeast SIR2 proteins restored growth of SIR2-deficient S. enterica on acetate and propionate, suggesting that eukaryotic cells may also use SIR2 proteins to control the synthesis of acetyl-CoA by the level of acetylation of acetyl-CoA synthetases. Consistent with this idea, growth of a quintuple sir2 hst1 hst2 hst3 hst4 mutant strain of the yeast Saccharomyces cerevisiae on acetate or propionate was severely impaired. The data suggest that the Hst3 and Hst4 proteins are the most important for allowing growth on these short-chain fatty acids.     

Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli

Sirtuins are NAD+-dependent protein deacetylase enzymes that are broadly conserved from bacteria to human, and have been implicated to play important roles in gene regulation, metabolism and longevity. cobB is a bacterial sirtuin that deacetylates acetyl-CoA synthetase (Acs) at an active site lysine to stimulate its enzymatic activity. Here, we report the structure of cobB bound to an acetyl-lysine containing non-cognate histone H4 substrate. A comparison with the previously reported archaeal and eukaryotic sirtuin structures reveals the greatest variability in a small zinc-binding domain implicated to play a particularly important role in substrate-specific binding by the sirtuin proteins. Comparison of the cobB/histone H4 complex with other sirtuin proteins in complex with acetyl-lysine containing substrates, further suggests that contacts to the acetyl-lysine side-chain and beta-sheet interactions with residues directly C-terminal to the acetyl-lysine represent conserved features of sirtuin-substrate recognition. Isothermal titration calorimetry studies were used to compare the affinity of cobB for a variety of cognate and non-cognate acetyl-lysine-bearing peptides revealing an exothermic reaction with relatively little discrimination between substrates. In contrast, similar studies employing intact acetylated Acs protein as a substrate reveal a binding reaction that is endothermic, suggesting that cobB recognition of substrate involves a burial of hydrophobic surface and/or structural rearrangement involving substrate regions distal to the acetyl-lysine-binding site. Together, these studies suggest that substrate-specific binding by sirtuin proteins involves contributions from the zinc-binding domain of the enzyme and substrate regions distal to the acetyl-lysine-binding site.     

Structure of Sir2Tm bound to a propionylated peptide

Lysine propionylation is a recently identified post‐translational modification that has been observed in proteins such as p53 and histones and is thought to play a role similar to acetylation in modulating protein activity. Members of the sirtuin family of deacetylases have been shown to have depropionylation activity, although the way in which the sirtuin catalytic site accommodates the bulkier propionyl group is not clear. We have determined the 1.8 Å structure of a Thermotoga maritima sirtuin, Sir2Tm, bound to a propionylated peptide derived from p53. A comparison with the structure of Sir2Tm bound to an acetylated peptide shows that hydrophobic residues in the active site shift to accommodate the bulkier propionyl group. Isothermal titration calorimetry data show that Sir2Tm binds propionylated substrates more tightly than acetylated substrates, but kinetic assays reveal that the catalytic rate of Sir2Tm deacylation of propionyl‐lysine is slightly reduced to acetyl‐lysine. These results serve to broaden our understanding of the newly identified propionyl‐lysine modification and the ability of sirtuins to depropionylate, as well as deacetylate, substrates.     

The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization

Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism.     

Characterization of acetyl-CoA synthetase kinetics and ATP-binding

BackgroundThe superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo.MethodsIn this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry.ResultsThe results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although k_cat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding.ConclusionsThe differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated.General SignificanceThe results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.     

Conserved metabolic regulatory functions of sirtuins

Silent information regulator 2 (Sir2) proteins, or sirtuins, are protein deacetylases/mono-ADP-ribosyltransferases found in organisms ranging from bacteria to humans. Their dependence on nicotinamide adenine dinucleotide (NAD+) links their activity to cellular metabolic status. In bacteria, the sirtuin CobB regulates the metabolic enzyme acetyl-coenzyme A (acetyl-CoA) synthetase. The earliest function of sirtuins therefore may have been regulation of cellular metabolism in response to nutrient availability. Recent findings support the idea that sirtuins play a pivotal role in metabolic control in higher organisms, including mammals. This review surveys evidence for an emerging role of sirtuins as regulators of metabolism in mammals.