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1.
Summary With the use of acetone extraction, reversed-phase High-Performance Liquid Chromatography and fluorimetric monitoring, the quantity of coenzyme F420 in mixed liquors and rumen contents can be measured. A synthetic analog of coenzyme F420 is used as an internal standard to compensate for differences in fluorimetric monitoring. The method allows the detection of one picomol of coenzyme F420 and the differentiation between different forms of the coenzyme known to be present in various methanogenic bacteria. 相似文献
2.
Summary Fluorimetric monitoring of potential methanogenic activity (coenzyme F420) of bacterial community from methane digesters is until now resstricted to a few particular cases because severe interference frequnetly occurs, or the concentration is too low when digesting highly diluted wastes. A method of purification and/or concentration, based on purification of the cellular fraction of the sample by centrifugation and washing, is proposed. In biomethanation of spent liquor from citric acid fermentation, a typical case where interference largely obscures the F420 fluorescence signal, precision is shown to be better than ± 5% (confidence interval [t.05 (3)]) for F420 concentration down to 200 nM, and the detection limit is below 10 nM. 相似文献
3.
Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80°C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF430, the prosthetic group of methylcoenzyme M reductase, F560, an oxidation product of this compound, coenzyme F420, F342, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF430, coenzyme F420, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied. 相似文献
4.
Summary The relationship between the coenzyme F420 content and the activity of methanogenic microorganisms was investigated under different cultivation conditions in anaerobic reactors. The coenzyme F420 concentration depends on the substrate used and the cultivation conditions. Coenzyme F420 appears not to be a measure of the total methanogenic activity but rather a measure of the amount of methanogenic microorganisms in mixed anaerobic cultures. 相似文献
5.
Relationship of Intracellular Coenzyme F420 Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri 总被引:3,自引:2,他引:1
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The use of F420 as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H2 and CO2, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F420 was followed by high-resolution fluorescence spectroscopy. F420 concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein−1; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g−1 depending on growth conditions. The content of F420 in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F420 content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F420 approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F420 content. However, qCH4(F420), calculated by dividing the methane production rate by the coenzyme F420 concentration, almost paralleled qCH4(protein). These results suggest that F420 may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH4(F420) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity. 相似文献
6.
Methane-forming archaea are strictly anaerobic microbes and are essential for global carbon fluxes since they perform the terminal step in breakdown of organic matter in the absence of oxygen. Major part of methane produced in nature derives from the methyl group of acetate. Only members of the genera Methanosarcina and Methanosaeta are able to use this substrate for methane formation and growth. Since the free energy change coupled to methanogenesis from acetate is only − 36 kJ/mol CH4, aceticlastic methanogens developed efficient energy-conserving systems to handle this thermodynamic limitation. The membrane bound electron transport system of aceticlastic methanogens is a complex branched respiratory chain that can accept electrons from hydrogen, reduced coenzyme F420 or reduced ferredoxin. The terminal electron acceptor of this anaerobic respiration is a mixed disulfide composed of coenzyme M and coenzyme B. Reduced ferredoxin has an important function under aceticlastic growth conditions and novel and well-established membrane complexes oxidizing ferredoxin will be discussed in depth. Membrane bound electron transport is connected to energy conservation by proton or sodium ion translocating enzymes (F420H2 dehydrogenase, Rnf complex, Ech hydrogenase, methanophenazine-reducing hydrogenase and heterodisulfide reductase). The resulting electrochemical ion gradient constitutes the driving force for adenosine triphosphate synthesis. Methanogenesis, electron transport, and the structure of key enzymes are discussed in this review leading to a concept of how aceticlastic methanogens make a living. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. 相似文献
7.
Detlef Schulze Martina Menkhaus Rainer Fiebig Hanswerner Dellweg 《Applied microbiology and biotechnology》1988,29(5):506-510
Summary Anaerobic treatment of gelatine-containing model waste water and baker's yeast manufacturing effluent was investigated in upflow anaerobic sludge blanket (UASB) reactors. During start up a correlation between coenzyme F
420 content and methane production in the reactor was observed. By monitoring coenzyme F
420 concentrations a certain prediction of methanogenic activities was possible. 相似文献
8.
Fluorimetric monitoring of methanogenesis in anaerobic digestors 总被引:2,自引:0,他引:2
A new parameter, QCH4(F420), is proposed to determine the potential methanogenic activity in the mixed microbial communities of anaerobic digestors. It is based on the particular fluorimetric properties of F420, a coenzyme common to and specific for methanogenic bacteria. 相似文献
9.
S. Rospert J. Breitung K. Ma B. Schwörer C. Zirngibl R. K. Thauer D. Linder R. Huber K. O. Stetter 《Archives of microbiology》1991,156(1):49-55
Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H2 and CO2 and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two -, - and -subunits and that it contained the nickel porphinoid coenzyme F430 as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the -subunit was determined. A comparison with the N-terminal sequences of the -subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity.Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO2 (apparent Vmax at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N
5,N10-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N
5,N10-methylenetetrahydromethanopterin dehydrogenase (H2-forming), 33 U/mg; N
5,N10-methylenetetrahydromethanopterin reductase (coenzyme F420 dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F420-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent Km values for these enzymes and the effect of salts on their activities were determined.The coenzyme F420 present in M. kandleri was identified as coenzyme F420-2 with 2 -glutamyl residues.Abbreviations H–S-CoM
coenzyme M
- CH3–S-CoM
methylcoenzyme M
- H–S-HTP
7-mercaptoheptanoylthreonine phosphate
- MFR
methanofuran
- CHO-MFR
formyl-MFR
- H4MPT
tetrahydromethanopterin
- CHO–H4MPT
N
5-formyl-H4MPT
- CH=H4MPT+
N
5,N10-methenyl-H4MPT
- CH2=H4MPT
N
5,N10-methylene-H4MPT
- CH3–H4MPT
N
5-methyl-H4MPT
- F420
coenzyme F420
- 1 U=
1 mol/min 相似文献
10.
Summary A quantitative assay for analogues of coenzyme F420 is presented. The assay combines separation of the coenzymes by binary reversed-phase high performance liquid chromatography with detection by fluorescence, yielding high specificity and sensitivity. Quantification is by calibration with a coenzyme F420 standard or by employing coenzyme F420 fragments as internal standards. 相似文献
11.
《Journal of biotechnology》1986,4(6):325-332
Growth of Methanobacterium thermoautotrophicum could be followed by measuring the intensity of fluorescence directly in the culture vessel, avoiding conventional time-consuming extraction procedures of fluorescent coenzymes. The influence of light scattering by the bacteria was investigated. It could be shown, that light scattering had only little effect on the measurement of coenzyme F420 fluorescence. However, culture fluorescence did not correlate to methanogenic activity, due to superposition of bacterial fluorescence by fluorescence from cell-free coenzyme which accumulates in the culture medium. By use of time-resolved laser spectroscopy, different fluorescence lifetimes were obtained for intracellular (1.0 ns) and extracellular (2.5 ns) components, respectively. A combination of this technique with photobleaching measurements for direct determination of F420 content of bacteria in a culture is proposed. 相似文献
12.
Summary Real-time on-line fluorescence measurements of the coenzymes NAD(P)H and F420 were evaluated as indicators of stability in a glucose-fed anaerobic methanogenic digester. A probe designed forin situ fluorimetric measurement of NAD(P)H provided an assessment of activity of the total microbial community, while the response of a fluorescence probe designed to measure coenzyme F420 correlated well with methanogenic activity. The two fluorescence-monitoring probes responded directly to fermentation imbalance during periods of substrate overloading and corresponded to traditional offline measurements, suggesting that the probes may be suitable candidates for inclusion in an on-line process control system for anaerobic digestion.Florida Agricultural Experimental Station, Journal Series no. R-00326 相似文献
13.
A rapid, novel and cost-effective spectrofluorimetric method developed to determine moxifloxacin (MFX) in pharmaceutical preparations because MFX in a pH 10 medium could reduce the fluorescence intensity of l -tryptophan. The maximum fluorescence excitation and emission wavelengths were found to be 280 and 363 nm respectively. A range of factors affecting fluorescence quenching and the effect of co-existing substances were investigated. Fluorescence quenching values (ΔF = FL-tryptophan − FMoxi-L-tryptophan) displayed a strong linear relationship with the MFX concentration ranging from 0.2 to 8.0 μg/ml under optimum conditions. The limit of detection was found to be 6.1 × 10−4 μg/ml. The proposed method was shown to be suitable for MFX determination in pharmaceutical tablets and biological fluids by the linearity, recovery and limit of detection. The spectrofluorimetric approach that has been developed is extremely eco-friendly, as evidenced by the fact that all the experimental components and solvents were safe for the environment. 相似文献
14.
Jasper Kunow Beatrix Schwörer Karl O. Stetter Rudolf K. Thauer 《Archives of microbiology》1993,160(3):199-205
Archaeoglobus fulgidus, a sulfate-reducing Archaeon with a growth temperature optimum of 83°C, uses the 5-deazaflavin coenzyme F420 rather than pyridine nucleotides in catabolic redox processes. The organism does, however, require reduced pyridine nuclcotides for biosynthetic purposes. We describe here that the Archaeon contains a coenzyme F420-dependent NADP reductase which links anabolism to catabolism. The highly thermostable enzyme was purfied 3600-fold by affinity chromatography to apparent homogeneity in a 60% yield. The native enzyme with an apparent molecular mass of 55 kDa was composed of only one type of subunit of apparent molecular mass of 28 kDa. Spectroscopic analysis of the enzyme did not reveal the presence of any chromophoric prosthetic group. The purified enzyme catalyzed the reversible reduction of NADP (apparent K
M 40 M) with reduced F420 (apparent K
M 20M) with a specific activity of 660 U/mg (apparent V
max) at pH 8.0 (pH optimum) and 80°C (temperature optimum). It was specific for both coenzyme F420 and NADP. Sterochemical investigations showed that the F420-dependent NADP reductase was Si face specific with respect to C5 of F420 and Si face specific with respect to C4 of NADP.Abbreviations F420
coenzyme F420
- F420H2
1,5-dihydrocoenzyme F420
- H4MPT
tetrahydromethanopterin
- CH=H4MPT
N5, N10-methylenetetrahydromethanopterin
- MFR
methanofuran
- HPLC
high performance liquid chromatography
- methylene-H4MPT dehydrogenase
N5, N10-methylenetetrahydromethanopterin dehydrogenase
- 1 U =
1 mol/min 相似文献
15.
S. F. Baron D. S. Williams H. D. May P. S. Patel H. C. Aldrich J. G. Ferry 《Archives of microbiology》1989,151(4):307-313
The ultrastructural locations of the coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F420-dependent formate dehydrogenase activity or F420-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.Abbreviation PBST
Phosphate-buffered saline containing 0.1% (v/v) Triton X-100 相似文献
16.
Washed membranes prepared from H2+CO2- or formate-grown cells of Methanococcus voltae catalyzed the oxidation of coenzyme F420H2 and the reduction of the heterodisulfide (CoB–S–S–CoM) of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate,
which is the terminal electron acceptor of the methanogenic pathway. The reaction followed a 1:1 stoichiometry according to
the equation: F420H2 + COB–S–S–CoM → F420 + CoM–SH + CoB–SH. These findings indicate that the reaction depends on a membrane-bound F420H2-oxidizing enzyme and on the heterodisulfide reductase, which remains partly membrane-bound after cell lysis. To elucidate
the nature of the F420H2-oxidizing protein, washed membranes were solubilized with detergent, and the enzyme was purified by sucrose density centrifugation,
anion-exchange chromatography, and gel filtration. Several lines of evidence indicate that F420H2 oxidation is catalyzed by a membrane-associated F420-reducing hydrogenase. The purified protein catalyzed the H2-dependent reduction of methyl viologen and F420. The apparent molecular mass and the subunit composition (43, 37, and 27 kDa) are almost identical to those of the F420-reducing hydrogenase that has already been purified from Mc. voltae. Moreover, the N-terminus of the 37-kDa subunit is identical to the amino acid sequence deduced from the fruG gene of the operon encoding the selenium-containing F420-reducing hydrogenase from Mc. voltae. A distinct F420H2 dehydrogenase, which is present in methylotrophic methanogens, was not found in this organism.
Received: 18 September 1998 / Accepted: 2 November 1998 相似文献
17.
Meike Goenrich Evert C. Duin Felix Mahlert Rudolf K. Thauer 《Journal of biological inorganic chemistry》2005,10(4):333-342
Methyl-coenzyme M reductase (MCR) catalyses the formation of methane from methyl-coenzyme M (CH3-S-CoM) and coenzyme B (HS-CoB) in methanogenic archaea. The enzyme has an 222 subunit structure forming two structurally interlinked active sites each with a molecule F430 as a prosthetic group. The nickel porphinoid must be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-based electron paramagnetic resonance (EPR) signal and a UV–vis spectrum with an absorption maximum at 385 nm. This state is called the MCR-red1 state. In the presence of coenzyme M (HS-CoM) and coenzyme B the MCR-red1 state is in part converted reversibly into the MCR-red2 state, which shows a rhombic Ni(I)-based EPR signal and a UV–vis spectrum with an absorption maximum at 420 nm. We report here for MCR from Methanothermobacter marburgensis that the MCR-red2 state is also induced by several coenzyme B analogues and that the degree of induction by coenzyme B is temperature-dependent. When the temperature was lowered below 20°C the percentage of MCR in the red2 state decreased and that in the red1 state increased. These changes with temperature were fully reversible. It was found that at most 50% of the enzyme was converted to the MCR-red2 state under all experimental conditions. These findings indicate that in the presence of both coenzyme M and coenzyme B only one of the two active sites of MCR can be in the red2 state (half-of-the-sites reactivity). On the basis of this interpretation a two-stroke engine mechanism for MCR is proposed. 相似文献
18.
J. T. Keltjens J. M. H. Hermans G. J. F. A. Rijsdijk C. Van der Drift G. D. Vogels 《Antonie van Leeuwenhoek》1988,54(3):207-220
F430 is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F430 is thermolabile and in the presence of acetonitrile or C10
in4
sup-
two epimerization products are obtained upon heating; in the absence of these compounds F430 is oxidized to 12, 13-didehydro-F430. The latter is stereoselectively reduced under H2 atmosphere to F430 by cell-free extracts of M. barkeri or M. thermoautotrophicum. H2 may be replaced by the reduced methanogenic electron carrier coenzyme F420.Abbreviations CH3S-CoM
methylcoenzyme M, 2-methylthioethanesulfonic acid
- HS-CoM
coenzyme M, 2-mercaptoethanesulfonic acid
- F430
Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton
- 13-epi-F430 and 12,13-di-epi-F430
the 12, 13- and 12, 13-derivatives of F430
- 12, 13-didehydro-F430
F430 oxidized at C-12 and C-13
- coenzyme F420
7,8-didemethyl-8-hydroxy-5-deazaflavin derivative
- coenzyme F420H2
reduced coenzyme F420
- MV+
methylviologen semiquinone
- HPLC
high-performance liquid chromatography 相似文献
19.
Jan T. Keltjens William B. Whitman C. Gerda Caerteling Annette M. van Kooten Ralph S. Wolfe Godfried D. Vogels 《Biochemical and biophysical research communications》1982,108(2):495-503
2-Mercaptoethanesulfonic acid (coenzyme M), or a derivative of it, and a yellow chromophore, known as the nickel-containing tetrapyrrole factor F430, occur in the prosthetic group of methylcoenzyme M reductase in an equimolar amount, and bound to each other; this enzyme catalyzes the final step of methane production. The prosthetic group, which is called coenzyme MF430, was isolated from the purified enzyme and was extracted from cells. The presence of coenzyme M was confirmed by a bioassay using Methanobrevibacter ruminantium and by the use of chemical and physicochemical analyses. 相似文献
20.
Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis
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Ellene H Mashalidis Apostolos G Gittis Aurelie Tomczak Chris Abell Clifton E Barry David N Garboczi 《Protein science : a publication of the Protein Society》2015,24(5):729-740
Coenzyme F420 is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F420 plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F420 is important to Mtb redox metabolism, little is known about the enzymes that bind F420 and the reactions that they catalyze. We have identified a novel F420-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F420. The crystal structure of the complex of Rv1155 and F420 reveals one F420 molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F420 complex provide clues about its role in the bacterium. 相似文献