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Acetyl-CoA

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Stephen W. Ragsdale – One of the best experts on this subject based on the ideXlab platform.

  • Pulse-chase studies of the synthesis of Acetyl-CoA by carbon monoxide dehydrogenase/Acetyl-CoA synthase: evidence for a random mechanism of methyl and carbonyl addition.
    The Journal of biological chemistry, 2008
    Co-Authors: Javier Seravalli, Stephen W. Ragsdale

    Abstract:

    Carbon monoxide dehydrogenase/Acetyl-CoA synthase catalyzes Acetyl-CoA synthesis from CO, CoA, and a methylated corrinoid iron-sulfur protein, which acts as a methyl donor. This reaction is the last step in the Wood-Ljungdahl pathway of anaerobic carbon fixation. The binding sequence for the three substrates has been debated for over a decade. Different binding orders imply different mechanisms (i.e. paramagnetic versus diamagnetic mechanisms). Ambiguity arises because CO and CoA can each undergo isotopic exchange with Acetyl-CoA, suggesting that either of these two substrates could be the last to bind to the Acetyl-CoA synthase active site. Furthermore, carbonylation, CoA binding, and methyl transfer can all occur in the absence of the other two substrates. Here, we report pulse-chase studies, which unambiguously establish the order in which the three substrates bind. Although a CoA pulse is substantially diluted by excess CoA in the chase, isotope recovery of a pulse of labeled CO or methyl group is unaffected by the presence of excess unlabeled CO or methyl group in the chase. These results demonstrate that CoA is the last substrate to bind and that CO and the methyl group bind randomly as the first substrate in Acetyl-CoA synthesis. Up to 100% of the methyl groups and CoA and up to 60-70% of the CO employed in the pulse phase can be trapped in the product Acetyl-CoA.

  • Nickel-Containing Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase†,‡
    Chemical reviews, 1996
    Co-Authors: Stephen W. Ragsdale, Manoj Kumar

    Abstract:

    This article reviews an enzyme with two important catalytic activities, carbon monoxide dehydrogenase (CODH) (reaction 1) and Acetyl-CoA synthase (ACS) (reaction 2). These reactions are key to an autotrophic pathway that has become known as the reductive Acetyl-CoA or the Wood/Ljungdahl pathway. ACS also catalyzes two exchange reactions that have been valuable in elucidating the mechanism of Acetyl-CoA synthesis: an exchange reaction between CO and the carbonyl group of Acetyl-CoA (reaction 3) and an exchange reaction between free CoA and the CoA moiety of Acetyl-CoA (reaction 4). Ten years ago, one of the authors (Ragsdale) and Harland Wood, first proposed that the CODH from acetogenic bacteria catalyzes the final steps in Acetyl-CoA synthesis and, therefore, should be renamed Acetyl-CoA synthase. It was previously accepted that these steps occurred on a cobalt-containing corrinoid protein. This was a controversial proposal that required the existence of carbonyl, methyl, and acetyl enzyme adducts. Proof of this postulate was nontrivial since it required the characterization of enzyme-bound intermediates. With the goal of detecting and characterizing intermediates in the pathway, the authors` laboratory and others began to probe the enzyme in the resting state and at different stages of the catalytic cycle with sensitive spectroscopic methods.more » This review summarizes the fruits of the combined labor of the laboratories working on this interesting problem. 237 refs.« less

  • Enzymology of the Acetyl-CoA pathway of CO2 fixation.
    Critical reviews in biochemistry and molecular biology, 1991
    Co-Authors: Stephen W. Ragsdale

    Abstract:

    AbstractWe know of three routes that organisms have evolved to synthesize complex organic molecules from CO2: the Calvin cycle. the reverse tricarboxylic acid cycle, and the reductive Acetyl-CoA pathway. This review describes the enzymatic steps involved in the Acetyl-CoA pathway, also called the Wood pathway, which is the major mechanism of CO2 fixation under anaerobic conditions. The Acetyl-CoA pathway is also able to form Acetyl-CoA from carbon monoxide.There are two parts to the Acetyl-CoA pathway: (1) reduction of CO2 to methyltetrahydrofolate (methyl-H4folate) and (2) synthesis of Acetyl-CoA from methyl-H, folate, a carboxyl donor such as CO or CO2, and CoA. This pathway is unique in that the major intermediates are enzyme-bound and are often organometallic complexes. Our current understanding of the pathway is based on radioactive and stable isotope tracer studies, purification of the component enzymes (some extremely oxygen sensitive), and identification of the enzyme-bound intcrmediates by chroma…

Rudolf K Thauer – One of the best experts on this subject based on the ideXlab platform.

  • coupled ferredoxin and crotonyl coenzyme a coa reduction with nadh catalyzed by the butyryl coa dehydrogenase etf complex from clostridium kluyveri
    Journal of Bacteriology, 2008
    Co-Authors: Fuli Li, Julia Hinderberger, Henning Seedorf, Jin Zhang, Wolfgang Buckel, Rudolf K Thauer

    Abstract:

    Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (Acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by Acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the Acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of Acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0′ = -410 mV) with NADH (E0′ = -320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0′ = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.

  • Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri
    Journal of Bacteriology, 2007
    Co-Authors: Fuli Li, Julia Hinderberger, Henning Seedorf, Jin Zhang, Wolfgang Buckel, Rudolf K Thauer

    Abstract:

    Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (Acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by Acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the Acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of Acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0′ = −410 mV) with NADH (E0′ = −320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0′ = −10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.

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

  • Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica.
    Frontiers in Microbiology, 2017
    Co-Authors: Farshad Borjian, Peter Schonheit, Ulrike Johnsen, Ivan A. Berg

    Abstract:

    Growth on acetate or other Acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of Acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of Acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to β-methylmalyl-CoA. The cleavage of β-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another Acetyl-CoA molecule give rise to two C4-dicarboxylic acids, thus regenerating the initial Acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica, succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii, and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as characteristic enzymes of this cycle.

  • purification and characterization of two extremely thermostable enzymes phosphate acetyltransferase and acetate kinase from the hyperthermophilic eubacterium thermotoga maritima
    Journal of Bacteriology, 1999
    Co-Authors: Annekatrin Bock, Jurgen Glasemacher, Roland Schmidt, Peter Schonheit

    Abstract:

    Acetate is an important end product of energy-yielding fermentation processes of many anaerobic and facultative procaryotes. Generally acetate is formed from acetyl coenzyme A (Acetyl-CoA), a central intermediate of metabolism. The mechanism of conversion of Acetyl-CoA to acetate in prokaryotes, which is coupled with ATP formation, has recently been shown to be dependent on the phylogenetic domain to which the organisms belong (33, 34). (i) In all eubacteria analyzed, Acetyl-CoA is converted to acetate by the “classical” mechanism involving two enzymes, phosphate acetyltransferase (PTA) (EC 2.3.1.8) and acetate kinase (AK) (EC 2.7.2.1). ATP is formed in the acetate kinase reaction by the mechanism of substrate-level phosphorylation. Acetyl-CoA + Pi ⇌ acetyl phosphate + CoA (PTA) Acetyl phosphate + ADP ⇌ acetate + ATP (AK)

    (ii) In all acetate forming archaea studied so far, including anaerobic hyperthermophiles and aerobic mesophilic halophiles, the conversion of Acetyl-CoA to acetate and the formation of ATP from ADP and phosphate is catalyzed by only one enzyme, an Acetyl-CoA synthetase (ADP forming) (33, 34). Acetyl-CoA + ADP + Pi ⇌ acetate + ATP + CoA

    This unusual synthetase, which was first discovered in the anaeobic eukaryote Entamoeba histolytica (23, 30), is part of a novel mechanism of acetate formation and energy conservation in prokaryotes.

    Acetate also serves as substrate of catabolism and anabolism in several aerobic and anaerobic prokaryotes. The activation of acetate to Acetyl-CoA, which is the first step prior to its utilization in metabolism, is catalyzed either by a single enzyme, an AMP-forming Acetyl-CoA synthetase (EC 6.2.1.1) (acetate + CoA + ATP ⇌ Acetyl-CoA + AMP + PPi) or by the AK-PTA couple operating in the reverse direction as described above (12, 33, 36, 40). Besides their function in acetate metabolism, PTA and AK play a role, via acetyl phosphate, in various other processes. For example, in Escherichia coli, acetyl phosphate functions as the phosphoryl donor of response regulator proteins of two-component systems, and a function as a global regulatory signal has therefore been proposed (22, 44).

    To date, acetate kinases and phosphate acetyltransferases have been purified from various bacteria and from the archaeon Methanosarcina thermophila. However, these enzymes have not yet been isolated and characterized from hyperthermophilic prokaryotes, which are considered to represent the most ancient living organisms (39).

    We have recently studied the glucose metabolism of the hyperthermophilic Thermotoga maritima, (Toptimum = 80°C), which belongs to the deepest branches in the phylogenetic tree within the bacterial domain. The organism ferments glucose to acetate, CO2, H2, and various amounts of lactate (15, 35). Glucose degradation to pyruvate proceeds via the classical Embden-Meyerhof pathway, and pyruvate oxidation to Acetyl-CoA involves pyruvate:ferredoxin oxidoreductase. The conversion of Acetyl-CoA to acetate and ATP is catalyzed by PTA and AK (34, 35), which is the mechanism of acetate formation typical of bacteria (see above).

    In this communication we report on the purification and characterization of AK and PTA from the hyperthermophilic eubacterium Thermotoga maritima.

  • 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, 1997
    Co-Authors: Jurgen Glasemacher, Annekatrin Bock, Roland Schmid, Peter Schonheit

    Abstract:

    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 (