AMP-forming acetyl-CoA Synthetase Research Articles

Acetyl-CoA synthetase activity is enzymatically regulated by lysine acetylation using acetyl-CoA or acetyl-phosphate as donor molecule

The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuA•AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5′-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuA•AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuA•AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels.

Site-specific and kinetic characterization of enzymatic and nonenzymatic protein acetylation in bacteria

Reversible Nε-lysine acetylation has emerging as an important metabolic regulatory mechanism in microorganisms. Herein, we systematically investigated the site-specific and kinetic characterization of enzymatic (lysine acetyltransferase) and nonenzymatic acetylation (AcP-dependent or Acyl-CoA-dependent), as well as their different effect on activity of metabolic enzyme (AMP-forming acetyl-CoA synthetase, Acs). It was found that Bacillus subtilis acetyl-CoA synthetase (BsAcsA) can be acetylated in vitro either catalytically by lysine acetyltransferase BsAcuA and Ac-CoA (at low concentration), or nonenzymatically by Ac-CoA or AcP (at high concentration). Two distinct mechanisms show preference for different lysine acetylation site (enzymatic acetylation for K549 and nonenzymatic acetylation for K524), and reveal different dynamics of relative acetylation changes at these lysine sites. The results demonstrated that lysine residues on the same protein exhibit different acetylation reactivity with acetyl-phosphate and acetyl-CoA, which was determined by surface accessibility, three-dimensional microenvironment, and pKa value of lysine. Acetyl-CoA synthetase is inactivated by AcuA-catalyzed acetylation, but not by nonenzymatic acetylation.

Functional analysis of an AMP forming acetyl CoA synthetase from Leishmania donovani by gene overexpression and targeted gene disruption approaches

Leishmaniasis, a neglected tropical disease is endemic in 98 countries and >350 million people are at risk of getting the infection. The existing chemotherapy of Leishmaniasis is limited due to adverse effects, resistance to existing drugs and increasing cases of HIV-Leishmaniasis co-infection. Hence, there is a need to identify novel metabolic pathways for design of new chemical entities. Acetyl-CoA synthetase (AceCS) is an enzyme of acetate metabolic pathway whose functions are unknown in Leishmania parasite. AceCS from Leishmania donovani (LdAceCS) is significantly different from human host to be explored as a potential drug candidate to develop parasite specific inhibitors. To dissect the functions of LdAceCS in Leishmania promastigotes, two approaches were followed. LdAceCS overexpressing parasites were generated by episomal expression of LdAceCS in promastigotes and single knockout (SKO) cell lines of LdAceCS were generated by targeted gene disruption. An insight into the phenotypic changes undergone by the overexpressors revealed an increase in LdAceCS activity, total lipid content, infectivity and ergosterol levels by ~2.2, 2.2, 1.65 and 3 fold respectively with respect to wild type. Similarly SKO transgenic parasites exhibited ~2.5, 3, 1.5 and 3 fold decrease in activity, total lipid content, infectivity and ergosterol respectively. Repeated attempts to generate null mutants failed thus indicating that LdAceCS is essential for the parasite and can be selectively targeted to combat Leishmania infection. The present study demonstrates that LdAceCS is important for in vitro macrophage infection and is also essential for biosynthesis of total lipids and ergosterol.

Characterization and expression of AMP-forming Acetyl-CoA Synthetase from Dunaliella tertiolecta and its response to nitrogen starvation stress.

AMP-forming acetyl-CoA synthetase (ACS) catalyzes the formation of acetyl-CoA. Here, a cDNA of ACS from Dunaliella tertiolecta (DtACS) was isolated using RACEs. The full-length DtACS cDNA (GenBank: KT692941) is 2,464 bp with a putative ORF of 2,184 bp, which encodes 727 amino acids with a predicted molecular weight of 79.72 kDa. DtACS has a close relationship with Chlamydomonas reinhardtii and Volvox carteri f. nagariensis. ACSs existing in Bacteria, Archaea and Eukaryota share ten conserved motifs (A1–A10) and three signature motifs (I–III) of the acyl-adenylate/thioester forming enzyme superfamily. DtACS was expressed in E. coli BL21 as Trx-His-tagged fusion protein (~100 kDa) and the enzymatic activity was detected. The recombinant DtACS was purified by HisTrapTM HP affinity chromatography to obtain a specific activity of 52.873 U/mg with a yield of 56.26%, which approached the specific activity of ACS isolated from other eukaryotes. Kinetic analysis indicated that the Km of DtACS was 3.59 mM for potassium acetate, and the purified DtACS exhibited a temperature optimum of 37 °C and a pH optimum of 8.0. In addition, the expression levels of DtACS were increased after nitrogen starvation cultivation, indicating that ACS activity may be related to the lipid accumulation under nitrogen deficient condition.

Genome analysis and heterologous expression of acetate-activating enzymes in the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.Electronic supplementary materialThe online version of this article (doi:10.1007/s00203-012-0829-7) contains supplementary material, which is available to authorized users.

AMP-Forming Acetyl Coenzyme A Synthetase in the Outermost Membrane of the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of ∼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2) fixation.

Acetate activation in Methanosaeta thermophila: characterization of the key enzymes pyrophosphatase and acetyl-CoA synthetase.

The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PPi) and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (K M = 0.27 ± 0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell.

Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome

Acyl-coenzyme A synthetases (ACSs) catalyze the fundamental, initial reaction in fatty acid metabolism. "Activation" of fatty acids by thioesterification to CoA allows their participation in both anabolic and catabolic pathways. The availability of the sequenced human genome has facilitated the investigation of the number of ACS genes present. Using two conserved amino acid sequence motifs to probe human DNA databases, 26 ACS family genes/proteins were identified. ACS activity in either humans or rodents was demonstrated previously for 20 proteins, but 6 remain candidate ACSs. For two candidates, cDNA was cloned, protein was expressed in COS-1 cells, and ACS activity was detected. Amino acid sequence similarities were used to assign enzymes into subfamilies, and subfamily assignments were consistent with acyl chain length preference. Four of the 26 proteins did not fit into a subfamily, and bootstrap analysis of phylograms was consistent with evolutionary divergence. Three additional conserved amino acid sequence motifs were identified that likely have functional or structural roles. The existence of many ACSs suggests that each plays a unique role, directing the acyl-CoA product to a specific metabolic fate. Knowing the full complement of ACS genes in the human genome will facilitate future studies to characterize their specific biological functions.

Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase

AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site.

Disruption of the Operon Encoding Ehb Hydrogenase Limits Anabolic CO 2 Assimilation in the Archaeon Methanococcus maripaludis

Methanococcus maripaludis is a mesophilic archaeon that reduces CO2 to methane with H2 or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ehb. To determine the role of Ehb, a deletion in the ehb operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessary to restore growth to nearly wild-type levels, suggesting that Ehb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ehb in anabolic CO2 assimilation in methanococci.

AMP-forming acetyl-CoA synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation

Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate+ATP+CoA --> Acetyl-CoA+AMP+PP(i)). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41 degrees C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37 degrees C. Using the N-terminal amino acid sequence an open reading frame - coding for a 74 kDa protein - was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.

Regulation of acetate and acetyl-CoA converting enzymes during growth on acetate and/or glucose in the halophilic archaeon Haloarcula marismortui

Haloarcula marismortui formed acetate during aerobic growth on glucose and utilized acetate as growth substrate. On glucose/acetate mixtures diauxic growth was observed with glucose as the preferred substrate. Regulation of enzyme activities, related to glucose and acetate metabolism was analyzed. It was found that both glucose dehydrogenase (GDH) and ADP-forming acetyl-CoA synthetase (ACD) were upregulated during periods of glucose consumption and acetate formation, whereas both AMP-forming acetyl-CoA synthetase (ACS) and malate synthase (MS) were downregulated. Conversely, upregulation of ACS and MS and downregulation of ACD and GDH were observed during periods of acetate consumption. MS was also upregulated during growth on peptides in the absence of acetate. From the data we conclude that a glucose-inducible ACD catalyzes acetate formation whereas acetate activation is catalyzed by an acetate-inducible ACS; both ACS and MS are apparently induced by acetate and repressed by glucose.

Mechanisms of acetate formation and acetate activation in halophilic archaea

The halophilic archaea Halococcus (Hc.) saccharolyticus, Haloferax (Hf.) volcanii, and Halorubrum (Hr.) saccharovorum were found to generate acetate during growth on glucose and to utilize acetate as a growth substrate. The mechanisms of acetate formation from acetyl-CoA and of acetate activation to acetyl-CoA were studied. Hc. saccharolyticus, exponentially growing on complex medium with glucose, formed acetate and contained ADP-forming acetyl-CoA synthetase (ADP-ACS) rather than acetate kinase and phosphate acetyltransferase or AMP-forming acetyl-CoA synthetase. In the stationary phase, the excreted acetate was completely consumed, and cells contained AMP-forming acetyl-CoA synthetase (AMP-ACS) and a significantly reduced ADP-ACS activity. Hc. saccharolyticus, grown on acetate as carbon and energy source, contained only AMP-ACS rather than ADP-ACS or acetate kinase. Cell suspensions of Hc. saccharolyticus metabolized acetate only when they contained AMP-ACS activity, i.e., when they were obtained after growth on acetate or from the stationary phase after growth on glucose. Suspensions of exponential glucose-grown cells, containing only ADP-ACS but not AMP-ACS, did not consume acetate. Similar results were obtained for the phylogenetic distantly related halophilic archaea Hf. volcanii and Hf. saccharovorum. We conclude that, in halophilic archaea, the formation of acetate from acetyl-CoA is catalyzed by ADP-ACS, whereas the activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS.

Evolution of the AMP-forming acetyl-CoA synthetase gene in the Drosophilidae family.

Analysis of the AMP-forming ACS gene was performed in 12 species of the Drosophilidae family. Systematically four introns, aligned at the same positions, were detected, but none of them showed a position similar to those known for species outside the Drosophilidae family. The average length of introns varied from 63 to 75 bp but in two species Drosophila takahashii and D. kikkawai the length of the second intron was 343 and 210 bp, respectively. In coding regions, about 80% of the third codon positions were substituted while first and second positions showed, respectively, 14% and 6% substitutions. Interestingly, the divergence observed at the protein level between species was very low. The phylogenetic tree based on the DNA sequences of the exons was mainly in agreement with taxonomic classification and previous molecular phylogenies except for D. ananassae, which appeared more closely related to D. subobscura and D. funebris than to the species of the melanogaster group.

Mechanisms of acetate formation and acetate activation in halophilic archaea.

The halophilic archaea Halococcus (Hc.) saccharolyticus, Haloferax (Hf.) volcanii, and Halorubrum (Hr.) saccharovorum were found to generate acetate during growth on glucose and to utilize acetate as a growth substrate. The mechanisms of acetate formation from acetyl-CoA and of acetate activation to acetyl-CoA were studied. Hc. saccharolyticus, exponentially growing on complex medium with glucose, formed acetate and contained ADP-forming acetyl-CoA synthetase (ADP-ACS) rather than acetate kinase and phosphate acetyltransferase or AMP-forming acetyl-CoA synthetase. In the stationary phase, the excreted acetate was completely consumed, and cells contained AMP-forming acetyl-CoA synthetase (AMP-ACS) and a significantly reduced ADP-ACS activity. Hc. saccharolyticus, grown on acetate as carbon and energy source, contained only AMP-ACS rather than ADP-ACS or acetate kinase. Cell suspensions of Hc. saccharolyticus metabolized acetate only when they contained AMP-ACS activity, i.e., when they were obtained after growth on acetate or from the stationary phase after growth on glucose. Suspensions of exponential glucose-grown cells, containing only ADP-ACS but not AMP-ACS, did not consume acetate. Similar results were obtained for the phylogenetic distantly related halophilic archaea Hf. volcanii and Hf. saccharovorum. We conclude that, in halophilic archaea, the formation of acetate from acetyl-CoA is catalyzed by ADP-ACS, whereas the activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS.

Purification and properties of acetyl-CoA synthetase (ADP-forming), an archaeal enzyme of acetate formation and ATP synthesis, from the hyperthermophile Pyrococcus furiosus.

Acetyl-CoA synthetase (ADP-forming) is an enzyme in Archaea that catalyzes the formation of acetate from acetyl-CoA and couples this reaction with the synthesis of ATP from ADP and Pi (acetyl-CoA + ADP + Pi --> acetate + ATP + CoA) [Schifer, T., Selig, M. & Schonheit, P. (1993) Arch. Microbiol. 159, 72-83]. The enzyme from the anaerobic hyperthermophile Pyrococcus furiosus was purified 96-fold with a yield of 20% to apparent electrophoretic homogeneity. The oxygen-stable enzyme had an apparent molecular mass of 145 kDa and was composed of two subunits with apparent molecular masses of 47 kDa and 25 kDa, indicating an alpha2beta2 structure. The N-terminal amino acid sequences of both subunits were determined; they do not show significant identity to other proteins in databases. The purified enzyme catalyzed the reversible conversion of acetyl-CoA, ADP and Pi to acetate, ATP and CoA. The apparent Vmax value in the direction of acetate formation was 18 U/mg (55 degrees C), the apparent Km values for acetyl-CoA, ADP and Pi were 17 microM, 60 microM and 200 microM, respectively. ADP and Pi could not be replaced by AMP and PPi, defining the enzyme as an ADP-forming rather than an AMP-forming acetyl-CoA synthetase. The apparent Vmax value in the direction of acetyl-CoA formation was about 40 U/mg (55 degrees C), and the apparent Km values for acetate, ATP and CoA were 660 microM, 80 microM and 30 microM, respectively. The purified enzyme was not specific for acetyl-CoA or acetate, in addition to acetyl-CoA (100%), the enzyme accepts propionyl-CoA (110%) and butyryl-CoA (92%), and in addition to acetate (100%), the enzyme accepts propionate (100%), butyrate (92%), isobutyrate (79%), valerate (36%) and isovalerate (34%), indicating that the enzyme functions as an acyl-CoA synthetase (ADP-forming) with a broad substrate spectrum. Succinate, phenylacetate and indoleacetate did not serve as substrates for the enzyme (<3%). In addition to ADP (100%), GDP (220%) and IDP (250%) were used, and in addition to ATP (100%), GTP (210%) and ITP (320%) were used. Pyrimidine nucleotides were not accepted. The enzyme was dependent on Mg2+, which could be partly substituted by Mn2+ and Co2+. The pH optimum was pH 7. The enzyme has a temperature optimum at 90 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions. The enzyme was stabilized against heat inactivation by salts. In the presence of KCI (1 M), which was most effective, the enzyme did not loose activity after 2 h incubation at 100 degrees C.