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Archives of Microbiology Nov 1996Acidaminococcus fermentans is able to ferment glutamate to ammonia, CO2, acetate, butyrate, and H2. The molecular hydrogen (approximately 10 kPa; E' = -385 mV) stems...
Acidaminococcus fermentans is able to ferment glutamate to ammonia, CO2, acetate, butyrate, and H2. The molecular hydrogen (approximately 10 kPa; E' = -385 mV) stems from NADH generated in the 3-hydroxybutyryl-CoA dehydrogenase reaction (E degrees ' = -240 mV) of the hydroxyglutarate pathway. In contrast to growing cells, which require at least 5 mM Na+, a Na+-dependence of the H2-formation was observed with washed cells. Whereas the optimal glutamate fermentation rate was achieved already at 1 mM Na+, H2 formation commenced only at > 10 mM Na+ and reached maximum rates at 100 mM Na+. The acetate/butyrate ratio thereby increased from 2.0 at 1 mM Na+ to 3.0 at 100 mM Na+. A hydrogenase and an NADH dehydrogenase, both of which were detected in membrane fractions, are components of a model in which electrons, generated by NADH oxidation inside of the cytoplasmic membrane, reduce protons outside of the cytoplasmic membrane. The entire process can be driven by decarboxylation of glutaconyl-CoA, which consumes the protons released by NADH oxidation inside the cell. Hydrogen production commences exactly at those Na+ concentrations at which the electrogenic H+/Na+-antiporter glutaconyl-CoA decarboxylase is converted into a Na+/Na+ exchanger.
Topics: Biotin; Carboxy-Lyases; Clostridium; Culture Media; Fermentation; Glutamic Acid; Glutarates; Gram-Negative Anaerobic Bacteria; Hydrogen; Hydrogenase; Ions; NADH Dehydrogenase; Sodium
PubMed: 8929282
DOI: 10.1007/s002030050394 -
European Journal of Biochemistry Jun 1995(R)-2-Hydroxyglutaryl-CoA dehydratase (HgdAB) from Acidaminococcus fermentans catalyses the reversible dehydration of its substrate to glutaconyl-CoA. The enzyme has to...
(R)-2-Hydroxyglutaryl-CoA dehydratase (HgdAB) from Acidaminococcus fermentans catalyses the reversible dehydration of its substrate to glutaconyl-CoA. The enzyme has to be activated by ATP, MgCl2, and Ti(III)citrate by an activator protein (HgdC) that is present in the organism at very low concentrations. Cell-free extracts of a recombinant Escherichia coli strain, in which hgdC was expressed, contained the activator with a specific activity of up to 45 U'/mg protein (1 U' is the amount of activator required to generate 1 U dehydratase activity under standard assay conditions). The recombinant protein was purified 44-fold to a specific activity of 2000 U'/mg. It is a homodimer (gamma 2, 54 kDa) and contains 4 mol non-heme iron and 3 mol inorganic sulfur. Under air, the activator has a half-life of seconds and even under strict anaerobic conditions it is very unstable. The amino acid sequence of the activator shows similarities to the ATP-binding motifs of several kinases. The dehydratase component was purified from its natural source revealing a heterodimer (alpha beta, 100 kDa) that contains 4 mol non-heme iron, 4 mol inorganic sulfur, 0.3 mol riboflavin, and 1 mol FMN. A mechanism is proposed in which an iron-sulfur cluster or a flavin donates one electron to the thiolester of the substrate (R)-2-hydroxyglutaryl-CoA. The resulting ketyl may eliminate the adjacent hydroxyl group yielding an enoxy radical from which the beta-hydrogen is abstracted as a proton leading to the ketyl of glutaconyl-CoA. In the final step, the latter is oxidized to the product, whereby the reduced enzyme is regenerated. It is suggested that during the activation step, the electron of this cycle is fed into the enzyme by Ti(III)citrate and energized by hydrolysis of ATP; both functions are apparently catalysed by the activator. The enzyme remains in this activated state for several turnovers, which may explain the requirement of only catalytic amounts of ATP and substoichiometric amounts of activator (dehydratase/activator ratio approximately 200:1). The oxidants 4-nitrobenzoate, 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone or chloramphenicol (all at concentrations greater than or equal to 1 microM) may trap this electron resulting in a reversible, transient inactivation of the dehydratase.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Bacterial Proteins; Enzyme Activation; Gram-Negative Anaerobic Bacteria; Hydro-Lyases; Molecular Sequence Data; Oxidants; Sequence Homology, Amino Acid
PubMed: 7607244
DOI: No ID Found -
Microbiome Jan 2016
PubMed: 26801625
DOI: 10.1186/s40168-016-0149-2 -
FEBS Letters Aug 1993(R)-2-Hydroxyglutaryl-CoA dehydratase (HGDA/B) from Acidaminococcus fermentans requires an activator protein for activity. This activator (HGDC) has not yet been...
(R)-2-Hydroxyglutaryl-CoA dehydratase (HGDA/B) from Acidaminococcus fermentans requires an activator protein for activity. This activator (HGDC) has not yet been purified from its natural source due to its low concentration combined with an extreme sensitivity towards oxygen. Gene expression in Escherichia coli identified an open reading frame (780 bp) as the gene encoding HGDC. Dehydratase activity was stimulated at least tenfold by cell-free extracts of E. coli cells transformed with a plasmid carrying hgdC. On the chromosome the hgdC gene is located just before hgdA and hgdB.
Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Cloning, Molecular; Enzyme Activation; Escherichia coli; Genes, Bacterial; Hydro-Lyases; Molecular Sequence Data; Oligodeoxyribonucleotides; Open Reading Frames; Veillonellaceae
PubMed: 8365476
DOI: 10.1016/0014-5793(93)80247-r -
Reviews of Infectious Diseases 1984Anaerobic bacteria are part of the normal flora of mucous membranes and outnumber aerobic bacteria in the oral cavity and gastrointestinal tract. Anaerobes can be... (Review)
Review
Anaerobic bacteria are part of the normal flora of mucous membranes and outnumber aerobic bacteria in the oral cavity and gastrointestinal tract. Anaerobes can be isolated from pediatric patients with various infections when appropriate techniques for transportation and cultivation of samples are employed. Frequently anaerobes are isolated in combination with other facultative or aerobic bacteria. The genera or groups of anaerobes most frequently isolated from pyogenic infections in children are (in order of decreasing frequency) OFFteroides, Clostridium, gram-positive cocci, Fusobacterium, gram-positive rods (Eubacterium, Lactobacillus, Propionibacterium, Actinomyces, and Bifidobacterium), and gram-negative cocci (Veillonella and Acidaminococcus). Clostridium perfringens causes bacteremia and wound infections. Clostridium botulinum can produce a paralytic toxin that causes a lethal illness in adults and a paralytic syndrome in infants. Clostridium difficile can cause antibiotic-associated colitis or diarrhea. Bacteroides fragilis is most frequently involved in intraabdominal infections, infections of the female genital tract, subcutaneous abscesses, and bacteremia. Bacteroides melaninogenicus and Bacteroides oralis are the predominent anaerobes in orofacial infections and aspiration pneumonia. Fusobacterium species are pathogens in aspiration pneumonia, brain abscesses, and orofacial infections. Anaerobic gram-positive cocci can be recovered from all types of infections but predominate in respiratory tract and intra-abdominal infections. Recognition of the pathogenic qualities of the various anaerobic organisms can assist in their prompt identification and in the initiation of appropriate therapy.
Topics: Actinomyces; Bacteria, Anaerobic; Bacterial Infections; Bacteroides; Bacteroides Infections; Child; Child, Preschool; Clostridium; Clostridium Infections; Fusobacterium; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Peptococcaceae; Propionibacterium; Veillonella
PubMed: 6372028
DOI: 10.1093/clinids/6.supplement_1.s187 -
Plant Biotechnology Journal Aug 2017CRISPR-mediated genome editing using the Streptococcus pyogenes Cas9 enzyme is revolutionizing life science by providing new, precise, facile and high-throughput tools... (Review)
Review
CRISPR-mediated genome editing using the Streptococcus pyogenes Cas9 enzyme is revolutionizing life science by providing new, precise, facile and high-throughput tools for genetic modification by the specific targeting of double-strand breaks in the genome of hosts. Plant biotechnologists have extensively used the S. pyogenes Cas9-based system since its inception in 2013. However, there are still some limitations to its even broader usage in plants. Major restrictions, especially in agricultural biotechnology, are the currently unclear regulatory status of plants modified with CRISPR/Cas9 and the lack of suitable delivery methods for some plant species. Solutions to these limitations could come in the form of new variants of genome editing enzymes that have recently been discovered and have already proved comparable to or even better in performance than S. pyogenes CRISPR/Cas9 in terms of precision and ease of delivery in mammal cells. Although some of them have already been tested in plants, most of them are less well known in the plant science community. In this review, we describe the following new enzyme systems engineered for genome editing, transcriptional regulation and cellular imaging-C2c2 from L. shahii; Cas9 from F. novicida, S. aureus, S. thermophiles, N. meningitidis; Cpf1 from F. novicida, Acidaminococcus and Lachnospiraceae; nickase, split, enhanced and other Cas9 variants from S. pyogenes; catalytically inactive SpCas9 linked to various nuclease or gene-regulating domains-with an emphasis on their advantages in comparison with the broadly used SpCas9. In addition, we discuss new possibilities they offer in plant biotechnology.
Topics: Biotechnology; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; Models, Biological; Plants, Genetically Modified; Streptococcus pyogenes
PubMed: 28371222
DOI: 10.1111/pbi.12736 -
New Microbes and New Infections Jun 2024
PubMed: 38799905
DOI: 10.1016/j.nmni.2024.101246 -
Archives of Microbiology Mar 2003The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from ( R)-2-hydroxyglutaryl-CoA to (...
The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from ( R)-2-hydroxyglutaryl-CoA to ( E)-glutaconyl-CoA catalyzed by 2-hydroxyglutaryl-CoA dehydratase, a two-component enzyme system. The actual dehydration is mediated by component D, which contains 1.0 [4Fe-4S](2+) cluster, 1.0 reduced riboflavin-5'-phosphate and about 0.1 molybdenum (VI) per heterodimer. The enzyme has to be activated by the extremely oxygen-sensitive [4Fe-4S](1+/2+)-cluster-containing homodimeric component A, which generates Mo(V) by an ATP/Mg(2+)-induced one-electron transfer. Previous experiments established that the hydroquinone state of a flavodoxin (m=14.6 kDa) isolated from A. fermentans served as one-electron donor of component A, whereby the blue semiquinone is formed. Here we describe the isolation and characterization of an alternative electron donor from the same organism, a two [4Fe-4S](1+/2+)-cluster-containing ferredoxin (m=5.6 kDa) closely related to that from Clostridium acidiurici. The protein was purified to homogeneity and almost completely sequenced; the magnetically interacting [4Fe-4S] clusters were characterized by EPR and Mössbauer spectroscopy. The redox potentials of the ferredoxin were determined as -405 mV and -340 mV. Growth experiments with A. fermentans in the presence of different iron concentrations in the medium (7-45 microM) showed that flavodoxin is the dominant electron donor protein under iron-limiting conditions. Its concentration continuously decreased from 3.5 micromol/g protein at 7 microM Fe to 0.02 micromol/g at 45 microM Fe. In contrast, the concentration of ferredoxin increased stepwise from about 0.2 micromol/g at 7-13 microM Fe to 1.1+/-0.1 micromol/g at 17-45 microM Fe.
Topics: Amino Acid Sequence; Bacteria, Anaerobic; Clostridium; Culture Media; Electron Spin Resonance Spectroscopy; Electron Transport; Ferredoxins; Flavodoxin; Hydro-Lyases; Iron; Molecular Sequence Data; Oxidation-Reduction; Sequence Homology, Amino Acid
PubMed: 12610725
DOI: 10.1007/s00203-003-0517-8 -
The Journal of Biological Chemistry Feb 2014Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation...
Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.
Topics: Acidaminococcus; Biocatalysis; Butyryl-CoA Dehydrogenase; Crystallography, X-Ray; Electron Transport; Electron-Transferring Flavoproteins; Electrons; Electrophoresis, Polyacrylamide Gel; Ferredoxins; Flavin-Adenine Dinucleotide; Flavins; Kinetics; Models, Biological; Molecular Docking Simulation; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Spectrophotometry, Ultraviolet
PubMed: 24379410
DOI: 10.1074/jbc.M113.521013 -
FEBS Letters Jan 1995In the course of glutamate fermentation by Acidaminococcus fermentans glutaconate coenzyme A-transferase catalyzes the transfer of CoAS- from acetyl-CoA to...
In the course of glutamate fermentation by Acidaminococcus fermentans glutaconate coenzyme A-transferase catalyzes the transfer of CoAS- from acetyl-CoA to (R)-2-hydroxyglutarate, forming (R)-2-hydroxyglutaryl-CoA. Glutamate (E) 54 of the beta-subunit was postulated to be directly involved in catalysis by formation of a CoASH ester intermediate [(1994) Eur. J. Biochem., in press]. In order to prove this preliminary result, the following mutations, beta E54A, beta E64A, beta E54Q and beta E54D, were introduced by mismatch oligonucleotide priming. As expected, beta E54A was inactive (0.02% of the wild-type), whereas beta E64A and beta E54D were active, 30% and > 7%, respectively. However, no CoASH intermediate was detected in the latter mutant, indicating a change in the catalytic mechanism. The activity of the beta E54Q mutant increased from 1% to almost 100% upon incubation with acetyl-CoA and glutaconate at 37 degrees C within 40 h. Hence, the substrates induced the conversion of the mutant glutamine residue into the glutamate residue of the wild-type enzyme.
Topics: Base Sequence; Binding Sites; Catalysis; Cloning, Molecular; Coenzyme A-Transferases; DNA Primers; Enzyme Activation; Escherichia coli; Glutamic Acid; Gram-Negative Anaerobic Bacteria; Molecular Sequence Data; Mutagenesis, Site-Directed
PubMed: 7805881
DOI: 10.1016/0014-5793(94)01351-z