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Peter Schonheit - One of the best experts on this subject based on the ideXlab platform.
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Acetate formation in the photoheterotrophic bacterium Chloroflexus aurantiacus involves an archaeal type ADP-forming Acetyl-CoA Synthetase isoenzyme I
FEMS microbiology letters, 2013Co-Authors: Marcel Schmidt, Peter SchonheitAbstract: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 .
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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, 2008Co-Authors: Christopher Brasen, Marcel Schmidt, Joachim Grotzinger, Peter SchonheitAbstract: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:
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AMP-forming Acetyl-CoA Synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation
Extremophiles, 2005Co-Authors: Christopher Brasen, Peter SchonheitAbstract: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.
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Mechanisms of acetate formation and acetate activation in halophilic archaea
Archives of Microbiology, 2001Co-Authors: Christopher Brasen, Peter SchonheitAbstract: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.
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Purification and Properties of Acetyl-CoA Synthetase (ADP-forming), an Archaeal Enzyme of Acetate Formation and ATP Synthesis, from the Hyperthermophile Pyrococcus furiosus
European journal of biochemistry, 1997Co-Authors: Jürgen Glasemacher, Anne-katrin Bock, Roland Schmid, Peter SchonheitAbstract: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) [Schafer, 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 α2β2 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°C), the apparent Km values for Acetyl-CoA, ADP and P, were 17 μM, 60 μM and 200 μM, respectively. ADP and Pi could not be replaced by AMP and PPiv 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°C), and the apparent Km values for acetate, ATP and CoA were 660 μM, 80 μM and 30 μM, 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 (
Miklós Müller - One of the best experts on this subject based on the ideXlab platform.
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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, 2000Co-Authors: Lidya B. Sánchez, Michael Y. Galperin, Miklós MüllerAbstract: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.
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Cloning and sequencing of an Acetyl-CoA Synthetase (ADP-forming) gene from the amitochondriate protist, Giardia lamblia.
Gene, 1999Co-Authors: Lidya B. Sánchez, Hilary G. Morrison, Mitchell L. Sogin, Miklós MüllerAbstract: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.
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Purification and characterization of the acetate forming enzyme, acetyl‐CoA Synthetase (ADP‐forming) from the amitochondriate protist, Giardia lamblia
FEBS letters, 1996Co-Authors: Lidya B. Sánchez, Miklós MüllerAbstract: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.
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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, 2000Co-Authors: Lidya B. Sánchez, Michael Y. Galperin, Miklós MüllerAbstract: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.
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Cloning and sequencing of an Acetyl-CoA Synthetase (ADP-forming) gene from the amitochondriate protist, Giardia lamblia.
Gene, 1999Co-Authors: Lidya B. Sánchez, Hilary G. Morrison, Mitchell L. Sogin, Miklós MüllerAbstract: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.
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Purification and characterization of the acetate forming enzyme, acetyl‐CoA Synthetase (ADP‐forming) from the amitochondriate protist, Giardia lamblia
FEBS letters, 1996Co-Authors: Lidya B. Sánchez, Miklós MüllerAbstract: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.
Louis M M Mouterde - One of the best experts on this subject based on the ideXlab platform.
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application of acetyl coa Synthetase from methanothermobacter thermautotrophicus to non native substrates
Enzyme and Microbial Technology, 2019Co-Authors: Louis M M Mouterde, Jon D. StewartAbstract:Abstract The substrate selectivity of the Trp416Gly mutant of Methanothermobacter thermautotrophicus Acetyl-CoA Synthetase (Trp416Gly MT-ACS1) was explored. The goal was to identify its substrate range, particularly for functionalized carboxylic acid substrates that would allow post-synthesis functionalization of CoA thioesters or downstream products using metathesis or Click chemistry. Relative activities were determined by in situ formation of acyl-hydroxamate iron (III) complexes. Trp416Gly MT-ACS1 showed good activities for saturated straight chain carboxylic acids from C2 to C8, for ω-alkenyl straight chain carboxylic acids from C4 to C7 and for ω-alkynyl straight chain carboxylic acids from C5 to C7. Carboxylic acids showing ≥20% conversion in screening reactions were used in preparative conversions that completely consumed the added CoASH.
Cheryl Ingram-smith - One of the best experts on this subject based on the ideXlab platform.
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Investigating the mechanism of ADP-forming Acetyl-CoA Synthetase from the protozoan parasite Entamoeba histolytica.
FEBS letters, 2017Co-Authors: Cheryl P. Jones, Kirin Khan, Cheryl Ingram-smithAbstract:ADP-forming Acetyl-CoA Synthetase (ACD) catalyzes the interconversion of Acetyl-CoA and acetate. The related succinyl-CoA Synthetase follows a three-step mechanism involving a single phosphoenzyme, but a novel four-step mechanism with two phosphoenzyme intermediates was proposed for Pyrococcus ACD. Characterization of enzyme variants of Entamoeba ACD in which the two proposed phosphorylated His residues were individually altered revealed that only His252 is essential for enzymatic activity. Analysis of variants altered at two residues proposed to interact with the phosphohistidine loop that swings between distinct parts of the active site are consistent with a mechanism involving a single phosphoenzyme intermediate. Our results suggest ACDs with different subunit structures may employ slightly different mechanisms to bridge the span between active sites I and II.
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Role of motif III in catalysis by Acetyl-CoA Synthetase.
Archaea (Vancouver B.C.), 2012Co-Authors: Cheryl Ingram-smith, Jerry L. Thurman, Karen Zimowski, Kerry S. SmithAbstract:The acyl-adenylate-forming enzyme superfamily, consisting of acyl- and aryl-CoA Synthetases, the adenylation domain of the nonribosomal peptide Synthetases, and luciferase, has three signature motifs (I–III) and ten conserved core motifs (A1–A10), some of which overlap the signature motifs. The consensus sequence for signature motif III (core motif A7) in Acetyl-CoA Synthetase is Y-X-S/T/A-G-D, with an invariant fifth position, highly conserved first and fourth positions, and variable second and third positions. Kinetic studies of enzyme variants revealed that an alteration at any position resulted in a strong decrease in the catalytic rate, although the most deleterious effects were observed when the first or fifth positions were changed. Structural modeling suggests that the highly conserved Tyr in the first position plays a key role in active site architecture through interaction with a highly conserved active-site Gln, and the invariant Asp in the fifth position plays a critical role in ATP binding and catalysis through interaction with the 2′- and 3′-OH groups of the ribose moiety. Interactions between these Asp and ATP are observed in all structures available for members of the superfamily, consistent with a critical role in substrate binding and catalysis for this invariant residue.
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Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase†
Biochemistry, 2006Co-Authors: Cheryl Ingram-smith, Barrett I. Woods, Kerry S. SmithAbstract: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.
