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1.
Formylmethanofuran:tetrahydromethanopterin formyltransferase is an essential enzyme in the one-carbon metabolism of methanogenic and sulfate-reducing archaea and of methylotrophic bacteria. The enzyme, which is devoid of a prosthetic group, catalyzes the reversible formyl transfer between the two substrates coenzyme methanofuran and coenzyme tetrahydromethanopterin (H4MPT) in a ternary complex catalytic mechanism. The structure of the formyltransferase without its coenzymes has been determined earlier. We report here the structure of the enzyme in complex with both coenzymes at a resolution of 2.0 A. Methanofuran, characterized for the first time in an enzyme structure, is embedded in an elongated cleft at the homodimer interface and fixed by multiple hydrophobic interactions. In contrast, tetrahydromethanopterin is only weakly bound in a shallow and wide cleft that provides two binding sites. It is assumed that the binding of the bulky coenzymes induces conformational changes of the polypeptide in the range of 3A that close the H4MPT binding cleft and position the reactive groups of both substrates optimally for the reaction. The key residue for substrate binding and catalysis is the strictly conserved Glu245. Glu245, embedded in a hydrophobic region and completely buried upon tetrahydromethanopterin binding, is presumably protonated prior to the reaction and is thus able to stabilize the tetrahedral oxyanion intermediate generated by the nucleophilic attack of the N5 atom of tetrahydromethanopterin onto the formyl carbon atom of formylmethanofuran. 相似文献
2.
The formylmethanofuran:tetrahydromethanopterin formyltransferase (FTR) from Methanobacterium thermoautotrophicum delta H was cloned and its sequence was determined. The clone was contained on a 4.8-kilobase BamHI fragment of M. thermoautotrophicum DNA ligated into pBR329. When this fragment was subcloned into the phagemid pTZ18R, a functional enzyme was synthesized under control of the lac promoter. Sequence analysis revealed the presence of a ribosome binding site and a possible terminator structure. The absence of an identifiable promoter lends credibility to the open reading frame which is present 5' to ftr. The ftr gene encodes an acidic protein with a calculated molecular weight of 31,401. The sequence of FTR does not appear to be homologous to any other sequenced proteins, including proteins which use pterin substrates. 相似文献
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Jan T. Keltjens 《Antonie van Leeuwenhoek》1984,50(4):383-396
Methanogenic bacteria gain their energy for growth from the conversion of a number of simple carbon compounds to methane. With a few exceptions all species known to date are able to reduce CO2 at which hydrogen acts as the electron donor. The reduction of CO2 can formally be considered to proceed through the formyl, the formaldehyde and the methyl level of reduction. These C1-units do not occur as free intermediates, but they remain bound to a number of unique coenzymes during the process. In this paper a survey is given of the structures and functions of these compounds; it deals with methanopterin derivatives, carbon dioxide reduction (CDR) factor, factor F430 and coenzyme M derivatives. A model of the process of methanogenesis that integrates previous ones and that allocates a function to the various coenzymes is presented.This paper is adapted from a treatise by the same author, entitled Coenzymes of methanogenesis. that was awarded the Kluyver prize 1984 by the Netherlands Society for Microbiology. 相似文献
6.
Formylmethanofuran:tetrahydromethanopterin formyltransferase from the hyperthermophilic methanogenic Archaeon Methanopyrus kandleri (growth temperature optimum 98°C) was crystallized by vapor diffusion methods. Crystal form M obtained with 2-methyl-2,4-pentanediol as precipitant displayed the space group P21 with unit cell parameters of a = 87.0 Å, b = 75.4 Å, c = 104.7 Å, and β = 113.9° and diffracted better than 2 Å resolution. Crystal form P grown from polyethylene glycol 8000 belonged to the space group I4122 and had unit cell parameters of 157.5 Å and 242.1 Å. Diffraction data to 1.73 Å were recorded. Crystal form S which was crystallized from (NH4)2SO4in the space group I4122 with unit cell parameters of 151.3 Å and 249.5 Å diffracted at least to 2.2 Å resolution. All crystal forms probably have four molecules per asymmetric unit and are suitable for X-ray structure analysis. © 1996 Wiley-Liss, Inc. 相似文献
7.
Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified from methanol grown Methanosarcina barkeri to apparent homogeneity and characterized with respect to its molecular and kinetic properties. The enzyme was found to be very similar to the formyltransferase from H2/CO2 grown Methanobacterium thermoautotrophicum. It also catalyzed the formation of N5-formyltetrahydromethanopterin rather than of N10-formyltetrahydromethanopterin from formylmethanofuran and tetrahydromethanopterin. 相似文献
8.
G. Hansson 《Applied microbiology and biotechnology》1979,6(4):351-359
Summary A mixed microbial culture, enriched from sewage sludge, was cultivated on a glucose medium in a high turbulence fermentor at constant temperature, pH and loading rate. Effects of high pCO2 (0.95 atm) and pCH4 (0.90 atm) were compared to those of low pCO2 (0.05 atm) and pCH4 (0.05 atm). Generally, rapid in creases in pCO2 resulted in rapid decreases in methane production and, conversely rapid decreases in pCO2 by N2-sparging resulted in rapid increases in methane production. Decreased values of methane production were also accompanied by acetic acid accumulation. No inhibiting effects of high pCH4 were detected. A method to obtain low pCO2 by gas recycling combined with CO2-absorption is proposed. 相似文献
9.
J Breitung G B?rner S Scholz D Linder K O Stetter R K Thauer 《European journal of biochemistry》1992,210(3):971-981
N-Formylmethanofuran(CHO-MFR):tetrahydromethanopterin(H4MPT) formyltransferase (formyltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100-fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N-terminal amino acid sequence of the polypeptide was determined. The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half-maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revealed that phosphates and sulfates both affected the Vmax rather than the Km for CHO-MFR and H4MPT. At the optimal salt concentration and at 65 degrees C, the Vmax was 2700 U/mg (1 U = 1 mumol/min), the Km for CHO-MFR was 50 microM and the Km for H4MPT was 100 microM. At 90 degrees C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65 degrees C. Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90 degrees C decreased in the order K2HPO4 = (NH4)2SO4 > KCl = NH4Cl = NaCl > Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary-complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus. 相似文献
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B. Schwörer J. Breitung A. R. Klein K. O. Stetter R. K. Thauer 《Archives of microbiology》1993,159(3):225-232
The sulfate-reducing Archaeoglobus fulgidus contains a number of enzymes previously thought to be unique for methanogenic Archaea. The purification and properties of two of these enzymes, of formylmethanofuran: tetrahydromethanopterin formyltransferase and of N
5,N
10-methylenetetrahydromethanopterin dehydrogenase (coenzyme F420 dependent) are described here. A comparison of the N-terminal amino acid sequences and of other molecular properties with those of the respective enzymes from three methanogenic Archaea revealed a high degree of similarity.Abbreviations H4MPT
tetrahydromethanopterin
- F420
coenzyme
- F420
formyltransferase, formylmethanofuran: tetrahydromethanopterin formyltransferase
- methylene-H4MPT dehydrogenase
N
5,N
10-methylenetetrahydromethanopterin dehydrogenase
- methylene-H4MPT recductase
N
5,N
10-methylenetetrahydromethanopterin reductase
- cyclohydrolase
N
5,N
10-methenyltetrahydromethanopterin cyclohydrolase
- APS
adenosine 5-phosphosulfate
- MOPS
3-(N-morpholino) propane sulfonic acid
- TRICINE
N-tris(hydroxymethyl)methylglycine
- MES
morpholinoethanesulfonic acid
- 1 U
1 mol/min 相似文献
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A fraction previously isolated from acid-treated supernatant fraction of Methanobacterium thermoautotrophicum by DEAE-Sephadex chromatography [Sauer, Mahadevan & Erfle (1984) Biochem. J. 221, 61-97] which was absolutely required for methane synthesis, has been separated into two compounds, tetrahydromethanopterin (H4MPT) and an as-yet-unidentified cofactor we call 'cytoplasmic cofactor'. H4MPT was identified by its u.v. spectrum and by 13C- and 1H-n.m.r. spectroscopy. The reduction of 2-(methylthio)ethanesulphonic acid (CH3-S-CoM) to methane by the membrane fraction from M. thermoautotrophicum was completely dependent on the addition of cytoplasmic cofactor. Methane synthesis from CO2, however, was only partially dependent on cofactor addition, and 57% of the original activity was retained in its absence. The kinetics of 14C labelling were consistent with the scheme methyl-H4MPT----CH3-S-CoM----methane, as has been proposed. This is the first time that direct experimental evidence has been presented to show that the proposed methyl transfer from H4MPT to coenzyme M (HS-CoM) actually occurs. 相似文献
15.
R. A. Berner 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1998,353(1365):75-82
A model (GEOCARB) of the long-term, or multimillion year, carbon cycle has been constructed which includes quantitative treatment of (1) uptake of atmospheric CO2 by the weathering of silicate and carbonate rocks on the continents, and the deposition of carbonate minerals and organic matter in oceanic sediments; and (2) the release of CO2 to the atmosphere via the weathering of kerogen in sedimentary rocks and degassing resulting from the volcanic-metamorphic-diagenetic breakdown of carbonates and organic matter at depth. Sensitivity analysis indicates that an important factor affecting CO2 was the rise of vascular plants in the Palaeozoic. A large Devonian drop in CO2 was brought about primarily by the acceleration of weathering of silicate rock by the development of deeply rooted plants in well-drained upland soils. The quantitative effect of this accelerated weathering has been crudely estimated by present-day field studies where all factors affecting weathering, other than the presence or absence of vascular plants, have been held relatively constant. An important additional factor, bringing about a further CO2 drop into the Carboniferous and Permian, was enhanced burial of organic matter in sediments, due probably to the production of microbially resistant plant remains (e.g. lignin). Phanerozoic palaeolevels of atmospheric CO2 calculated from the GEOCARB model generally agree with independent estimates based on measurements of the carbon isotopic composition of palaeosols and the stomatal index for fossil plants. Correlation of CO2 levels with estimates of palaeoclimate suggests that the atmospheric greenhouse effect has been a major factor in controlling global climate over the past 600 million years. 相似文献
16.
The effect of CO2 concentration on growth and glucose fermentation of Bacteroides fragilis was studied in a defined mineral medium. Batch culture experiments were done in closed tubes containing CO2 concentrations ranging from 10% to 100% (with appropriate amounts of bicarbonate added to maintain the pH at 6.7). These experiments revealed that CO2 had no influence on growth rate or cell yield when the CO2 concentration was above 30% CO2 (minimum available CO2–HCO
3
-
, 25.5 mM), whereas a slight decrease in these parameters was observed at 20% and 10% CO2 (available CO2–HCO
3
-
, 17 and 8.5 mM, respectively). If CO2–HCO
3
-
concentrations were below 10 mM, the lag phase lengthened and a decrease in maximal growth rate and cell yield were observed. The amount of acetate made decreased, while d-lactate concentration increased. A net production of CO2 allowed growth under conditions of extremely low concentrations of added CO2.When B. fragilis was grown in continuous culture with 100% CO2 or 100% N2, the dilution rate influenced the concentrations of acetate, succinate, propionate, d-lactate, l-malate and formate formed. Decreasing the dilution rate favored propionate and acetate production under both conditions. When the organism was grown with 100% N2, the amount of propionate formed was greater than the amount of succinate formed at all dilution rates. Except at slow dilution rates the reverse was true when 100% CO2 was used. B. fragilis was unable to grow at dilution rates faster than 0.154 h-1 when grown with 100% N2; the Y
glc
max
was 67.9 g DW cells/mol glucose and m
s was 0.064 mmol glucose/g DW·h. If the gas atmosphere was 100% CO2 the organism was washed out of the culture when the dilution rate exceeded 0.38 h-1; the Y
glc
max
was 59.4 g DW cells/mol glucose and m
s was 0.094 mmol glucose/g DW·h.Measurement of the phosphoenolpyruvate (PEP) carboxykinase (E.C. 4.1.1.49) with whole, permeabilized cells of B. fragilis showed an increase of specific enzyme activity with decreasing CO2 concentrations. The mechanisms used by B. fragilis to adjust to low levels of CO2 are discussed. 相似文献
17.
The magnitude and role of dark CO2 fixation were examined in nodules of intact soybean plants (Harosoy 63 × Rhizobium japonicum strain USDA 16). The estimated rate of nodule dark CO2 fixation, based on a 2 minute pulse-feed with 14CO2 under saturating conditions, was 102 micromoles per gram dry weight per hour. This was equivalent to 14% of net nodule respiration. Only 18% of this CO2 fixation was estimated to be required for organic and amino acid synthesis for growth and export processes. The major portion (75-92%) of fixed label was released as CO2 within 60 minutes. The labeling pattern during pulse-chase experiments was consistent with CO2 fixation by phosphoenolpyruvate carboxylase. During the chase, the greatest loss of label occurred in organic acids. Exposure of nodulated roots to Ar:O2 (80:20) did not affect dark CO2 fixation, while exposure to O2:CO2 (95:5) resulted in 54% inhibition. From these results, it was concluded that at least 66% of dark CO2 fixation in soybean may be involved with the production of organic acids, which when oxidized would be capable of providing at least 48% of the requirement for ATP equivalents to support nitrogenase activity. 相似文献
18.
Carbon dioxide interacts both with reactive nitrogen species and reactive oxygen species. In the presence of superoxide, NO reacts to form peroxynitrite that reacts with CO2 to give nitrosoperoxycarbonate. This compound rearranges to nitrocarbonate which is prone to further reactions. In an aqueous environment, the most probable reaction is hydrolysis producing carbonate and nitrate. Thus the net effect of CO2 is scavenging of peroxynitrite and prevention of nitration and oxidative damage. However, in a nonpolar environment of membranes, nitrocarbonate undergoes other reactions leading to nitration of proteins and oxidative damage. When NO reacts with oxygen in the absence of superoxide, a nitrating species N2O3 is formed. CO2 interacts with N2O3 to produce a nitrosyl compound that, under physiological pH, is hydrolyzed to nitrous and carbonic acid. In this way, CO2 also prevents nitration reactions. CO2 protects superoxide dismutase against oxidative damage induced by hydrogen peroxide. However, in this reaction carbonate radicals are formed which can propagate the oxidative damage. It was found that hypercapnia in vivo protects against the damaging effects of ischemia or hypoxia. Several mechanisms have been suggested to explain the protective role of CO2 in vivo. The most significant appears to be stabilization of the iron-transferrin complex which prevents the involvement of iron ions in the initiation of free radical reactions. 相似文献
19.
The role of carbon dioxide in light-activated hydrogen production by Chlamydomonas reinhardtii 总被引:1,自引:0,他引:1
Light-activated hydrogen and oxygen evolution as a function of CO2 concentration in helium were measured for the unicellular green alga Chlamydomonas reinhardtii. The concentrations were 58, 30, 0.8 and 0 ppm CO2. The objective of these experiments was to study the differential affinity of CO2/HCO
3
-
for their respective Photosystem II and Calvin cycle binding sites vis-à-vis photoevolution of molecular oxygen and the competitive pathways of hydrogen photoevolution and CO2 photoassimilation. The maximum rate of hydrogen evolution occurred at 0.8 ppm CO2, whereas the maximum rate of oxygen evolution occurred at 58 ppm CO2. The key result of this work is that the rate of photosynthetic hydrogen evolution can be increased by, at least partially, satisfying the Photosystem II CO2/HCO
3
-
binding site requirement without fully activating the Calvin-Benson CO2 reduction pathway. Data are presented which plot the rates of hydrogen and oxygen evolution as functions of atmospheric CO2 concentration in helium and light intensity. The stoichiometric ratio of hydrogen to oxygen changed from 0.1 at 58 ppm to approximately 2.5 at 0.8 ppm. A discussion of partitioning of photosynthetic reductant between the hydrogen/hydrogenase and Calvin-Benson cycle pathways is presented.Abbreviations PET
photosynthetic electron transport
- PS
Photosystem 相似文献
20.
Tetrahydromethanopterin, a carbon carrier in methanogenesis 总被引:20,自引:0,他引:20
J C Escalante-Semerena K L Rinehart R S Wolfe 《The Journal of biological chemistry》1984,259(15):9447-9455
Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented. 相似文献