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Acetyl-CoA Synthetase

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Peter Schonheit – One of the best experts on this subject based on the ideXlab platform.

  • Acetate formation in the photoheterotrophic bacterium Chloroflexus aurantiacus involves an archaeal type ADP-forming Acetyl-CoA Synthetase isoenzyme I
    FEMS microbiology letters, 2013
    Co-Authors: Marcel Schmidt, Peter Schonheit

    Abstract:

    The bacterium Chloroflexus aurantiacus excreted significant amounts of acetate during photohetero trophic growth on glucose and in resting cell suspensions. Up to 1.5 mol acetate per mol glucose were formed. In acetate-forming cells, the activities of phosphotransacetylase and acetate kinase, usually involved in acetate formation in Bacteria , could not be detected; instead, the cells contained an Acetyl-CoA Synthetase (ADP-forming) (ACD) (Acetyl-CoA + ADP + Pi → acetate + ATP + CoA), an enzyme so far reported in prokaryotes to be specific for acetate-forming Archaea . ACD, which was induced 10-fold during growth on glucose, was purified and the encoding gene was identified as Caur_3920 . The recombinant enzyme, a homotetrameric 300-kDa protein composed of 75-kDa subunits, was characterized as functional ACD. Substrate specificities and kinetic constants for Acetyl-CoA/acetate and other acyl-CoA esters/acids were determined, showing similarity of the C. aurantiacus ACD to archaeal ACD I isoenzymes, which are involved in acetate formation from sugars. This is the first report of a functional ACD involved in acetate formation in the domain of Bacteria .

  • reaction mechanism and structural model of adp forming acetyl coa Synthetase from the hyperthermophilic archaeon pyrococcus furiosus evidence for a second active site histidine residue
    Journal of Biological Chemistry, 2008
    Co-Authors: Christopher Brasen, Marcel Schmidt, Joachim Grotzinger, Peter Schonheit

    Abstract:

    Abstract In Archaea, acetate formation and ATP synthesis from Acetyl-CoA is catalyzed by an unusual ADP-forming Acetyl-CoA Synthetase (ACD) (Acetyl-CoA + ADP + Pi ⇆ acetate + ATP + HS-CoA) catalyzing the formation of acetate from Acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA Synthetases, which also include succinyl-CoA Synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the β-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (α2β2) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of Acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257α, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257α to ADP via transient phosphorylation of a second conserved histidine residue in theβ-subunit, His-71β. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues:

  • AMP-forming Acetyl-CoA Synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation
    Extremophiles, 2005
    Co-Authors: Christopher Brasen, Peter Schonheit

    Abstract:

    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°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°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.

Miklós Müller – One of the best experts on this subject based on the ideXlab platform.

  • Acetyl-CoA Synthetase from the amitochondriate eukaryote Giardia lamblia belongs to the newly recognized superfamily of acyl-CoA Synthetases (Nucleoside diphosphate-forming).
    The Journal of biological chemistry, 2000
    Co-Authors: Lidya B. Sánchez, Michael Y. Galperin, Miklós Müller

    Abstract:

    Abstract The gene coding for the Acetyl-CoA Synthetase (ADP-forming) from the amitochondriate eukaryote Giardia lamblia has been expressed in Escherichia coli. The recombinant enzyme exhibited the same substrate specificity as the native enzyme, utilizing Acetyl-CoA and adenine nucleotides as preferred substrates and less efficiently, propionyl- and succinyl-CoA. N- and C-terminal parts of the G. lamblia Acetyl-CoA Synthetase sequence were found to be homologous to the α- and β-subunits, respectively, of succinyl-CoA Synthetase. Sequence analysis of homologous enzymes from various bacteria, archaea, and the eukaryote, Plasmodium falciparum, identified conserved features in their organization, which allowed us to delineate a new superfamily of acyl-CoA Synthetases (nucleoside diphosphate-forming) and its signature motifs. The representatives of this new superfamily of thiokinases vary in their domain arrangement, some consisting of separate α- and β-subunits and others comprising fusion proteins in α-β or β-α orientation. The presence of homologs of Acetyl-CoA Synthetase (ADP-forming) in such human pathogens as G. lamblia, Yersinia pestis, Bordetella pertussis,Pseudomonas aeruginosa, Vibrio cholerae,Salmonella typhi, Porphyromonas gingivalis, and the malaria agent P. falciparum suggests that they might be used as potential drug targets.

  • Cloning and sequencing of an Acetyl-CoA Synthetase (ADP-forming) gene from the amitochondriate protist, Giardia lamblia.
    Gene, 1999
    Co-Authors: Lidya B. Sánchez, Hilary G. Morrison, Mitchell L. Sogin, Miklós Müller

    Abstract:

    A Giardia lamblia gene, Glacs, was cloned, sequenced and expressed in Escheria Coli. This gene codes for a 726 residue long Acetyl-CoA Synthetase (ADP-forming). This enzyme is responsible for the formation of acetate, a metabolic endproduct of G. lamblia. It is known from only two Type I amitochondriate eukaryotes, G. lamblia and Entamoeba histolytica and from the archaebacterium, Pyrococcus furiosus. With Glacs as query, homologous unidentified open reading frames were detected in the complete genomes of only a few archaebacteria and eubacteria. These form a new protein family present in all three domains of life, which probably plays a central role in the acyl-CoA metabolism but is of restricted taxonomic distribution.

  • Purification and characterization of the acetate forming enzyme, acetyl‐CoA Synthetase (ADP‐forming) from the amitochondriate protist, Giardia lamblia
    FEBS letters, 1996
    Co-Authors: Lidya B. Sánchez, Miklós Müller

    Abstract:

    Giardia lamblia, an amitochondriate eukaryote, contains Acetyl-CoA Synthetase (ADP-forming), an enzyme known only from one other eukaryote (Entamoeba histolytica) and a few anaerobic prokaryotes. The enzyme has been purified about 350-fold. The activity in the direction of acetate formation was dependent on ADP and inorganic phosphate. The reverse reaction could not be detected. Succinyl-CoA, propionyl-CoA and dADP were utilized with lower efficiency. The enzyme did not utilize AMP plus PPi thus differs from the broadly distributed Acetyl-CoA Synthetase (AMP-forming). The enzyme is responsible for acetate production accompanied by ATP generation, thus plays an important role in G. lamblia metabolism.

Lidya B. Sánchez – One of the best experts on this subject based on the ideXlab platform.

  • Acetyl-CoA Synthetase from the amitochondriate eukaryote Giardia lamblia belongs to the newly recognized superfamily of acyl-CoA Synthetases (Nucleoside diphosphate-forming).
    The Journal of biological chemistry, 2000
    Co-Authors: Lidya B. Sánchez, Michael Y. Galperin, Miklós Müller

    Abstract:

    Abstract The gene coding for the Acetyl-CoA Synthetase (ADP-forming) from the amitochondriate eukaryote Giardia lamblia has been expressed in Escherichia coli. The recombinant enzyme exhibited the same substrate specificity as the native enzyme, utilizing Acetyl-CoA and adenine nucleotides as preferred substrates and less efficiently, propionyl- and succinyl-CoA. N- and C-terminal parts of the G. lamblia Acetyl-CoA Synthetase sequence were found to be homologous to the α- and β-subunits, respectively, of succinyl-CoA Synthetase. Sequence analysis of homologous enzymes from various bacteria, archaea, and the eukaryote, Plasmodium falciparum, identified conserved features in their organization, which allowed us to delineate a new superfamily of acyl-CoA Synthetases (nucleoside diphosphate-forming) and its signature motifs. The representatives of this new superfamily of thiokinases vary in their domain arrangement, some consisting of separate α- and β-subunits and others comprising fusion proteins in α-β or β-α orientation. The presence of homologs of Acetyl-CoA Synthetase (ADP-forming) in such human pathogens as G. lamblia, Yersinia pestis, Bordetella pertussis,Pseudomonas aeruginosa, Vibrio cholerae,Salmonella typhi, Porphyromonas gingivalis, and the malaria agent P. falciparum suggests that they might be used as potential drug targets.

  • Cloning and sequencing of an Acetyl-CoA Synthetase (ADP-forming) gene from the amitochondriate protist, Giardia lamblia.
    Gene, 1999
    Co-Authors: Lidya B. Sánchez, Hilary G. Morrison, Mitchell L. Sogin, Miklós Müller

    Abstract:

    A Giardia lamblia gene, Glacs, was cloned, sequenced and expressed in Escheria Coli. This gene codes for a 726 residue long Acetyl-CoA Synthetase (ADP-forming). This enzyme is responsible for the formation of acetate, a metabolic endproduct of G. lamblia. It is known from only two Type I amitochondriate eukaryotes, G. lamblia and Entamoeba histolytica and from the archaebacterium, Pyrococcus furiosus. With Glacs as query, homologous unidentified open reading frames were detected in the complete genomes of only a few archaebacteria and eubacteria. These form a new protein family present in all three domains of life, which probably plays a central role in the acyl-CoA metabolism but is of restricted taxonomic distribution.

  • Purification and characterization of the acetate forming enzyme, acetyl‐CoA Synthetase (ADP‐forming) from the amitochondriate protist, Giardia lamblia
    FEBS letters, 1996
    Co-Authors: Lidya B. Sánchez, Miklós Müller

    Abstract:

    Giardia lamblia, an amitochondriate eukaryote, contains Acetyl-CoA Synthetase (ADP-forming), an enzyme known only from one other eukaryote (Entamoeba histolytica) and a few anaerobic prokaryotes. The enzyme has been purified about 350-fold. The activity in the direction of acetate formation was dependent on ADP and inorganic phosphate. The reverse reaction could not be detected. Succinyl-CoA, propionyl-CoA and dADP were utilized with lower efficiency. The enzyme did not utilize AMP plus PPi thus differs from the broadly distributed Acetyl-CoA Synthetase (AMP-forming). The enzyme is responsible for acetate production accompanied by ATP generation, thus plays an important role in G. lamblia metabolism.