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Butyryl-CoA

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Wolfgang Buckel – 1st expert on this subject based on the ideXlab platform

  • Reduction of ferredoxin or oxygen by flavin‐based electron bifurcation in Megasphaera elsdenii
    FEBS Journal, 2015
    Co-Authors: Nilanjan Pal Chowdhury, Jörg Kahnt, Wolfgang Buckel

    Abstract:

    Over 50 years ago, it was reported that, in the anaerobic rumen bacterium Megasphaera elsdenii, the reduction of crotonyl-CoA to Butyryl-CoA by NADH involved an electron transferring flavoprotein (Etf) as mediator [Baldwin RL, Milligan LP (1964) Biochim Biophys Acta 92, 421–432]. Purification and spectroscopic characterization revealed that this Etf contained 2 FAD, whereas, in the Etfs from aerobic and facultative bacteria, one FAD is replaced by AMP. Recently we detected a similar system in the related anaerobe Acidaminococcus fermentans that differed in the requirement of additional ferredoxin as electron acceptor. The whole process was established as flavin-based electron bifurcation in which the exergonic reduction of crotonyl-CoA by NADH mediated by Etf + Butyryl-CoA dehydrogenase (Bcd) was coupled to the endergonic reduction of ferredoxin also by NADH. In the present study, we demonstrate that, under anaerobic conditions, Etf + Bcd from M. elsdenii bifurcate as efficiently as Etf + Bcd from A. fermentans. Under the aerobic conditions used in the study by Baldwin and Milligan and in the presence of catalytic amounts of crotonyl-CoA or Butyryl-CoA, however, Etf + Bcd act as NADH oxidase producing superoxide and H2O2, whereas ferredoxin is not required. We hypothesize that, during bifurcation, oxygen replaces ferredoxin to yield superoxide. In addition, the formed Butyryl-CoA is re-oxidized by a second oxygen molecule to crotonyl-CoA, resulting in a stoichiometry of 2 NADH consumed and 2 H2O2 formed. As a result of the production of reactive oxygen species, electron bifurcation can be regarded as an Achilles’ heel of anaerobes when exposed to air.

  • effect of an oxygen tolerant bifurcating butyryl coenzyme a dehydrogenase electron transferring flavoprotein complex from clostridium difficile on butyrate production in escherichia coli
    Journal of Bacteriology, 2013
    Co-Authors: Wolfgang Buckel, E A Aboulnaga, Olaf Pinkenburg, Johannes Schiffels, Ahmed Elrefai, Thorsten Selmer

    Abstract:

    ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.

  • Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli.
    Journal of Bacteriology, 2013
    Co-Authors: E A Aboulnaga, Wolfgang Buckel, Olaf Pinkenburg, Johannes Schiffels, Ahmed A. El-refai, Thorsten Selmer

    Abstract:

    ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.

Thorsten Selmer – 2nd expert on this subject based on the ideXlab platform

  • effect of an oxygen tolerant bifurcating butyryl coenzyme a dehydrogenase electron transferring flavoprotein complex from clostridium difficile on butyrate production in escherichia coli
    Journal of Bacteriology, 2013
    Co-Authors: Wolfgang Buckel, E A Aboulnaga, Olaf Pinkenburg, Johannes Schiffels, Ahmed Elrefai, Thorsten Selmer

    Abstract:

    ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.

  • Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli.
    Journal of Bacteriology, 2013
    Co-Authors: E A Aboulnaga, Wolfgang Buckel, Olaf Pinkenburg, Johannes Schiffels, Ahmed A. El-refai, Thorsten Selmer

    Abstract:

    ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.

  • acryloyl coa reductase from clostridium propionicum an enzyme complex of propionyl coa dehydrogenase and electron transferring flavoprotein
    FEBS Journal, 2003
    Co-Authors: Marc Hetzel, Matthias Brock, Thorsten Selmer, Antonio J Pierik, BERNARD THOMAS GOLDING, Wolfgang Buckel

    Abstract:

    Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow–greenish enzyme complex [(α2βγ)4; molecular mass 600 ± 50 kDa] composed of a propionyl-CoA dehydrogenase (α2, 2 × 40 kDa) and an electron-transferring flavoprotein (ETF; β, 38 kDa; γ, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per α2βγ subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (Km = 2 ± 1 µm; kcat = 4.5 s−1 at 100 µm NADH) is 3-buten-2-one (methyl vinyl ketone; Km = 1800 µm; kcat = 29 s−1 at 300 µm NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (Km = 50 µm; kcat = 2.0 s−1) or Butyryl-CoA (Km = 100 µm; kcat = 3.5 s−1) as electron donor and 200 µm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of Butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the β and γ subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloyl-CoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.

E A Aboulnaga – 3rd expert on this subject based on the ideXlab platform

  • effect of an oxygen tolerant bifurcating butyryl coenzyme a dehydrogenase electron transferring flavoprotein complex from clostridium difficile on butyrate production in escherichia coli
    Journal of Bacteriology, 2013
    Co-Authors: Wolfgang Buckel, E A Aboulnaga, Olaf Pinkenburg, Johannes Schiffels, Ahmed Elrefai, Thorsten Selmer

    Abstract:

    ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.

  • Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli.
    Journal of Bacteriology, 2013
    Co-Authors: E A Aboulnaga, Wolfgang Buckel, Olaf Pinkenburg, Johannes Schiffels, Ahmed A. El-refai, Thorsten Selmer

    Abstract:

    ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.