Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli.

Acetyl coenzyme A synthetase (Acs) activates acetate to acetyl coenzyme A through an acetyladenylate intermediate; two other enzymes, acetate kinase (Ack) and phosphotransacetylase (Pta), activate acetate through an acetyl phosphate intermediate. We subcloned acs, the Escherichia coli open reading frame purported to encode Acs (F. R. Blattner, V. Burland, G. Plunkett III, H. J. Sofia, and D. L. Daniels, Nucleic Acids Res. 21:5408-5417, 1993). We constructed a mutant allele, delta acs::Km, with the central 0.72-kb BclI-BclI portion of acs deleted, and recombined it into the chromosome. Whereas wild-type cells grew well on acetate across a wide range of concentrations (2.5 to 50 mM), those deleted for acs grew poorly on low concentrations (< or = 10 mM), those deleted for ackA and pta (which encode Ack and Pta, respectively) grew poorly on high concentrations (> or = 25 mM), and those deleted for acs, ackA, and pta did not grow on acetate at any concentration tested. Expression of acs from a multicopy plasmid restored growth to cells deleted for all three genes. Relative to wild-type cells, those deleted for acs did not activate acetate as well, those deleted for ackA and pta displayed even less activity, and those deleted for all three genes did not activate acetate at any concentration tested. Induction of acs resulted in expression of a 72-kDa protein, as predicted by the reported sequence. This protein immunoreacted with antiserum raised against purified Acs isolated from an unrelated species, Methanothrix soehngenii. The purified E. coli Acs then was used to raise anti-E. coli Acs antiserum, which immunoreacted with a 72-kDa protein expressed by wild-type cells but not by those deleted for acs. When purified in the presence, but not in the absence, of coenzyme A, the E. coli enzyme activated acetate across a wide range of concentrations in a coenzyme A-dependent manner. On the basis of these and other observations, we conclude that this open reading frame encodes the acetate-activating enzyme, Acs.     

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.     

Regulation of acetyl coenzyme A synthetase in Escherichia coli

Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of acs and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce acs, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways.     

Alterations in membrane fluidity and dynamics in experimental colon cancer and its chemoprevention by diclofenac

Acs2p is one of two acetyl-coenzyme A synthetases in Saccharomyces cerevisiae. We have prepared and characterized a monoclonal antibody specific for Acs2p and find that Acs2p is localized primarily to the nucleus, including the nucleolus, with a minor amount in the cytosol. We find that Acs2p is required for replicative longevity: an acs2? strain has a reduced replicative life span compared to wild-type and acs1? strains. Furthermore, replicatively aged acs2? cells contain elevated levels of extrachromosomal rDNA circles, and silencing at the rDNA locus is impaired in an acs2? strain. These findings indicate that Acs2p-mediated synthesis of acetyl-CoA in the nucleus functions to promote rDNA silencing and replicative longevity in yeast.     

Global regulatory mutations in csrA and rpoS cause severe central carbon stress in Escherichia coli in the presence of acetate

The csrA gene encodes a small RNA-binding protein, which acts as a global regulator in Escherichia coli and other bacteria (T. Romeo, Mol. Microbiol. 29:1321-1330, 1998). Its key regulatory role in central carbon metabolism, both as an activator of glycolysis and as a potent repressor of glycogen biosynthesis and gluconeogenesis, prompted us to examine the involvement of csrA in acetate metabolism and the tricarboxylic acid (TCA) cycle. We found that growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media (e.g., tryptone broth). Cultures grown in the presence of >/=25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. Several hypotheses were examined to provide further insight into the effects of acetate on growth and metabolism in these strains. We determined that csrA positively regulates acs (acetyl-coenzyme A synthetase; Acs) expression and isocitrate lyase activity without affecting key TCA cycle enzymes or phosphotransacetylase. TCA cycle intermediates or pyruvate, but not glucose, galactose, or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. We conclude that central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism apparently are combined to cause metabolic stress by depleting the TCA cycle.     

Identification of two acyl-CoA synthetases from Pseudomonas putida GPo1: one is located at the surface of polyhydroxyalkanoates granules

Pseudomonas putida GPo1 is able to accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules as storage materials. PHA granules were isolated and analyzed for protein activities. An acyl-CoA-synthetase (ACS1) activity was detected from the purified PHA granules. The corresponding gene acs1 was then cloned from P. putida GPo1. With the genomic walking technique, a homologue acs2 located upstream of acs1 was discovered and cloned. Fusions of both acs1 and acs2 with the gene encoding the green fluorescent protein (GFP) were constructed and expressed in GPo1. In vivo fluorescence microscopy studies showed that the fluorescence generated from the ACS1-GFP was mainly associated with the PHA granules, whereas that from ACS2-GFP was mainly with the membrane of the cells. In the control strain (containing GFP alone) fluorescence was distributed evenly in the cytoplasm. We concluded that ACS1 is located on the PHA granules and may play a central role in mobilization of PHA, for example, conversion of hydroxycarboxylic acid monomers to hydroxycarboxyl-CoA, which can be further utilized by the cells.     

acs1 of Haemophilus influenzae type a capsulation locus region II encodes a bifunctional ribulose 5-phosphate reductase- CDP-ribitol pyrophosphorylase

The serotype-specific, 5.9-kb region II of the Haemophilus influenzae type a capsulation locus was sequenced and found to contain four open reading frames termed acs1 to acs4. Acs1 was 96% identical to H. influenzae type b Orf1, previously shown to have CDP-ribitol pyrophosphorylase activity (J. Van Eldere, L. Brophy, B. Loynds, P. Celis, I. Hancock, S. Carman, J. S. Kroll, and E. R. Moxon, Mol. Microbiol. 15:107-118, 1995). Low but significant homology to other pyrophosphorylases was only detected in the N-terminal part of Acs1, whereas the C-terminal part was homologous to several short-chain dehydrogenases/reductases, suggesting that Acs1 might be a bifunctional enzyme. To test this hypothesis, acs1 was cloned in an expression vector and overexpressed in Escherichia coli. Cells expressing this protein displayed both ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities, whereas these activities were not detectable in control cells. Acs1 was purified to near homogeneity and found to copurify with ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities. These had superimposable elution profiles from DEAE-Sepharose and Blue-Sepharose columns. The dehydrogenase activity was specific for ribulose 5-phosphate and NADPH in one direction and for ribitol 5-phosphate and NADP+ in the other direction and was markedly stimulated by CTP. The pyrophosphorylase showed activity with CTP and ribitol 5-phosphate or arabitol 5-phosphate. We conclude that acs1 encodes a bifunctional enzyme that converts ribulose 5-phosphate into ribitol 5-phosphate and further into CDP-ribitol, which is the activated precursor form for incorporation of ribitol 5-phosphate into the H. influenzae type a capsular polysaccharide.     

The role of acetyl-coenzyme a synthetase in Arabidopsis

The acs1 knockout mutant that has a disruption in the plastidic acetyl-coenzyme A (CoA) synthetase (ACS; At5g36880) gene was used to explore the role of this protein and plastidic acetate metabolism in Arabidopsis (Arabidopsis thaliana). Disruption of the ACS gene decreased ACS activity by 90% and largely blocked the incorporation of exogenous (14)C-acetate and (14)C-ethanol into fatty acids. Whereas the disruption had no significant effect on the synthesis of bulk seed triacylglycerols, the acs1 plants were smaller and flowered later. This suggests that the pyruvate dehydrogenase bypass provided by the aerobic fermentation pathway that converts pyruvate to acetate and probably on to fatty acids is important to the plants during normal growth. The role of ACS in destroying fermentative intermediates is supported by the increased sensitivity of the acs1 mutant to exogenous acetate, ethanol, and acetaldehyde compared to wild-type plants. Whereas these observations suggest that flux through the aerobic fermentation pathway is important, the reason for this flux is unclear. Interestingly, acetate is able to support high rates of plant growth on medium and this growth is blocked in the acs1 mutant.     

The chemotaxis response regulator CheY can catalyze its own acetylation

One of the processes by which CheY, the excitatory response regulator of chemotaxis in Escherichia coli, can be activated to generate clockwise flagellar rotation is by acetyl-CoA synthetase (Acs)-mediated acetylation. Deletion of Acs results in defective chemotaxis, indicating the involvement of Acs-mediated acetylation in chemotaxis. To investigate whether Acs is the sole acetylating agent of CheY, we purified the latter from a delta acs mutant. Mass spectrometry analysis revealed that this protein is partially acetylated in spite of the absence of Acs, suggesting that CheY can be post-translationally acetylated in vivo by additional means. Using [14C]AcCoA in the absence of Acs, we demonstrated that one of these means is autoacetylation, with AcCoA serving as an acetyl donor and with a rate similar to that of Acs-mediated acetylation. Biochemical characterization of autoacetylated CheY and mass spectrometry analysis of its tryptic digests revealed that its acetylated lysine residues are those found in CheY acetylated by Acs, but the acetylation-level distribution among the acetylation sites was different. Like CheY acetylated by Acs, autoacetylated CheY could be deacetylated by Acs. Also similarly to the case of Acs-mediated acetylation, the phosphodonors of CheY, CheA and acetyl phosphate, each inhibited the autoacetylation of CheY, whereas the phosphatase of CheY, CheZ, enhanced it. A reduced AcCoA level interfered with chemotaxis to repellents, suggesting that CheY autoacetylation may be involved in chemotaxis of E. coli. Interestingly, this interference was restricted to repellent addition and was not observed with attractant removal, thus endorsing our earlier suggestion that the signaling pathway triggered by repellent addition is not identical to that triggered by attractant removal.     

Comparative study of aromatic ring meta-cleavage enzymes in Pseudomonas strains with plasmid and chromosomal genetic control of the catabolism of biphenyl and m-toluate

It was shown that two different enzymes of aromatic ring oxidative meta-cleavage (2,3-dihydroxybiphenyl-1,2-dioxygenase), DBO and catechol-2,3-dioxygenase, C230) function in Pseudomonas strains with a plasmid and chromosomal genetic control of biphenyl and toluate catabolism. A comparative analysis of DBO's and C230's expressed by the pBS241 biphenyl degradative plasmid in P. putida BS893, pBS311 in P. putida U83, chromosomal genes in P. putida BF and C230 from P. putida PaW160 (pWWO) was carried out. It was found that the DBO's of all strains under study are highly specialized enzymes in respect of 2,3-dihydroxybiphenyl cleavage and are also able to cleave 3-methyl-catechol and catechol (but not 4-methylcatechol) at low rates. In contrast with DBO's, in Pseudomonas strains the substrate specificities of all C230's are variable. The C230's expressed by the D-plasmids pBS241 and pBC311 have a moderate affinity for catechol, 3-methyl- and 4-methylcatechol, but are unable to cleave 2,3-dihydroxybiphenyl. The C230 which is encoded by the chromosomal structure gene from P. putida BF is very similar to C230 which codes for the TOL-plasmid pWWO. These plasmid differ from C230's expressed by biphenyl D-plasmids due to their capability to cleave 2,3-dihydroxybiphenyl in addition to catechol cleavage. All DBO's and C230's under study possess a number of properties that are typical for the enzymes having an oxidative meta-cleaving effect. The different roles of these enzymes in biphenyl and toluate catabolism in Pseudomonas strains are discussed.     

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.     

Genetic and ultrastructural analysis of different mutants of Pseudomonas putida affected in the poly-3-hydroxy-n-alkanoate gene cluster

Functional analyses of the different proteins involved in the synthesis and accumulation of polyhydroxyalkanoates (PHAs) in P. putida U were performed using a mutant in which the pha locus had been deleted (PpUDeltapha). These studies showed that: (i) Pha enzymes cannot be replaced by other proteins in this bacterium, (ii) the transformation of PpDeltapha with a plasmid containing the locus pha fully restores the synthesis of bioplastics, (iii) the transformation of PpDeltapha with a plasmid harbouring the gene encoding the polymerase PhaC1 (pMCphaC1) permits the synthesis of polyesters (even in absence of phaC2ZDFI); however, in this strain (PpUDeltapha-pMCphaC1) the number of PHAs granules was higher than in the wild type, (iv) the expression of phaF in PpUDeltapha-pMCphaC1 restores the original phenotype, showing that PhaF is involved in the coalescence of the PHAs granules. Furthermore, the deletion of the phaDFI genes in P. putida U considerably decreases (> 70%) the biosynthesis of PHAs consisting of hydroxyalkanoates with aliphatic constituents, and completely prevents the synthesis of those ones containing aromatic monomers. Additional experiments revealed that the deletion of phaD in P. putida U strongly reduces the synthesis of PHA, this effect being restored by PhaF. Moreover, the overexpression of phaF in P. putida U, or in its DeltafadBA mutant, led to the collection of PHA over-producer strains.     

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.