Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.2.1.13 (acetyl-CoA synthetase)
451 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mode of action of carnitine on the growth of the yeast Torulopsis bovina ATCC 26014 was investigated. When 0.5-5 microM L-carnitine was added to the medium, the growth rate doubled for both aerobic and anaerobic cultures. Cells grown in the absence of carnitine contain 0.4 nmol of L-carnitine/g, wet weight, but with 5 microM L-carnitine in the media, cells contain 1400 nmol of carnitine/g, wet weight, by the end of exponential growth. When [1-14C]acetyl-L-carnitine was added to growth media, almost all of the radioactivity became cell-associated. Most of the 14C was incorporated into cell protein although considerable 14C was recovered in the fatty acid fraction of saponified cells. Analyses of the amino acids derived from radiolabeled protein showed that the acetyl[14C] of acetylcarnitine was in glutamate, arginine, proline, leucine, and lysine. In contrast, [1-14C]acetate labeled leucine and lysine. Isopycnic density gradient analysis demonstrated that carnitine acetyltransferase was primarily associated with mitochondria, while acetyl-CoA synthetase and acetyl-CoA hydrolase were cytosolic. Isolated mitochondria incorporated [14C]acetylcarnitine radioactivity into citrate and 2-oxoglutarate. The data are consistent with carnitine facilitating the transfer of acetyl groups from the cytosol into mitochondria for synthesis of citrate and its metabolites. These results demonstrate a role for carnitine in biosyntheses in the yeast T. bovina.
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PMID:A biosynthetic role for carnitine in the yeast Torulopsis bovina. 668 27

It is well established that the response regulator of the chemotaxis system of Escherichia coli, CheY, can undergo acetylation at lysine residues 92 and 109 via a reaction mediated by acetyl-CoA synthetase (Acs). The outcome is activation of CheY, which results in increased clockwise rotation. Nevertheless, it has not been known whether CheY acetylation is involved in chemotaxis. To address this question, we examined the chemotactic behaviour of two mutants, one lacking the acetylating enzyme Acs, and the other having an arginine-for-lysine substitution at residue 92 of CheY - one of the acetylation sites. The Deltaacs mutant exhibited much reduced sensitivity to chemotactic stimuli (both attractants and repellents) in tethering assays and greatly reduced responses in ring-forming, plug and capillary assays. Likewise, the cheY(92KR) mutant had reduced sensitivity to repellents in tethering assays and a reduced response in capillary assays. However, its response to the addition or removal of attractants was normal. These observations suggest that Acs-mediated acetylation of CheY is involved in chemotaxis and that the acetylation site Lys-92 is only involved in the response to repellents. The observation that, in the cheY(92KR) mutant, the addition of a repellent was not chemotactically equivalent to the removal of an attractant also suggests that there are different signalling pathways for attractants and repellents in E. coli.
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PMID:Acetylation of the response regulator, CheY, is involved in bacterial chemotaxis. 1135 78

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.
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PMID:The 1.75 A crystal structure of acetyl-CoA synthetase bound to adenosine-5'-propylphosphate and coenzyme A. 1262 52

Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.
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PMID:Fungal ammonia fermentation, a novel metabolic mechanism that couples the dissimilatory and assimilatory pathways of both nitrate and ethanol. Role of acetyl CoA synthetase in anaerobic ATP synthesis. 1472 82

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.
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PMID:Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli. 1501 90

Sir2 proteins form a family of NAD(+)-dependent protein deacetylases required for diverse biological processes, including transcriptional silencing, suppression of rDNA recombination, control of p53 activity, regulation of acetyl-CoA synthetase, and aging. Although structures of Sir2 enzymes in the presence and absence of peptide substrate or NAD(+) have been determined, the role of the enzyme in the mechanism of deacetylation and NAD(+) cleavage is still unclear. Here, we present additional structures of Sir2Af2 in several differently complexed states: in a productive complex with NAD(+), in a nonproductive NAD(+) complex with bound ADP-ribose, and in the unliganded state. We observe a new mode of NAD(+) binding that seems to depend on acetyl-lysine binding, in which the nicotinamide ring of NAD(+) is buried in the highly conserved "C" pocket of the enzyme. We propose a detailed structure-based mechanism for deacetylation and nicotinamide inhibition of Sir2 consistent with mutagenesis and enzymatic studies.
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PMID:Structural basis for the mechanism and regulation of Sir2 enzymes. 1502 35

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.
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PMID:Acetylation of the chemotaxis response regulator CheY by acetyl-CoA synthetase purified from Escherichia coli. 1532 42

AMP-forming acetyl-CoA synthetases (ACSs) are ubiquitous in all three domains of life. Here, we report the first characterization of an ACS from a hyperthermophilic organism, from the archaeon Pyrobaculum aerophilum. The recombinant ACS, the gene product of ORF PAE2867, showed extremely high thermostability and thermoactivity at temperatures around 100 degrees C. In contrast to known monomeric or homodimeric mesophilic ACSs, the P. aerophilum ACS was a 610 kDa homooctameric protein, with a significant lower content of thermolabile (Cys, Asn, and Gln) and higher content of charged (Glu, Lys, and Arg) amino acids. Kinetic analyses revealed an unusual broad substrate spectrum for organic acids and an extremely high affinity for acetate (K(m) 3 microM).
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PMID:A novel octameric AMP-forming acetyl-CoA synthetase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. 1564 62

Eleven strains of methanogenic bacteria were divided into two groups on the basis of the directionality (oxidative or reductive) of their citric acid pathways. These pathways were readily identified for most methanogens from the patterns of carbon atom labeling in glutamate, following growth in the presence of [2-C]acetate. All used noncyclic pathways, but members of the family Methanosarcinaceae were the only methanogens found to use the oxidative direction. Methanococcus jannaschii failed to incorporate carbon from acetate despite transmembrane equilibration comparable to other weak acids. This organism was devoid of detectable activities of the acetate-incorporating enzymes acetyl coenzyme A synthetase, acetate kinase, and phosphotransacetylase. However, incorporation of [1-C]-, [2-C]-, or [3-C]pyruvate during the growth of M. jannaschii was possible and resulted in labeling patterns indicative of a noncyclic citric acid pathway operating in the reductive direction to synthesize amino acids. Carbohydrates were labeled consistent with glucogenesis from pyruvate. Leucine, isoleucine, phenylalanine, lysine, formate, glycerol, and mevalonate were incorporated when supplied to the growth medium. Lysine was preferentially incorporated into the lipid fraction, suggesting a role as a phytanyl chain precursor.
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PMID:Metabolic Pathways in Methanococcus jannaschii and Other Methanogenic Bacteria. 1634 9

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.
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PMID:The chemotaxis response regulator CheY can catalyze its own acetylation. 1663 Jun 31


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