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R K Thauer

Publications and source records attributed to R K Thauer.

At least 19 recordsLinked to original sources
PubMed1992

Determination of the relative configuration of 5,6,7,8-tetrahydromethanopterin by two-dimensional NMR spectroscopy.

The relative configuration of the pterin moiety of 5,6,7,8-tetrahydromethanopterin 1, a coenzyme isolated from methanogenic archaea, has been determined by two-dimensional NMR spectroscopy of N5,N10-methenyl-5,6,7,8-tetrahydromethanopterin 2 to be rel-(6R; 7S; 11R). The complete proton resonance assignment of the pterin moiety of N5,N10-methylene-5,6,7,8-tetrahydromethanopterin 3 is described including the relative stereospecific assignment of the C(14a) methylene protons.

Magnetic Resonance Spectroscopy↗
PubMed1992

Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri.

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.

Amino Acid Sequence↗
PubMed1992

Substrate-analogue-induced changes in the nickel-EPR spectrum of active methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum.

Methyl-coenzyme-M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M [2-(methylthio)ethanesulfonate] and 7-mercaptoheptanoylthreonine phosphate in methanogenic archaea. The enzyme contains the nickel porphinoid coenzyme F430 as a prosthetic group. In the active, reduced (red) state, the enzyme displays two characteristic EPR signals, MCR-red1 and MCR-red2, probably derived from Ni(I). In the presence of the substrate methyl-coenzyme M, the rhombic MCR-red2 signal is quantitatively converted to the axial MCR-red1 signal. We report here on the effects of inhibitory substrate analogues on the EPR spectrum of the enzyme. 3-Bromopropanesulfonate (BrPrSO3), which is the most potent inhibitor of MCR known to date (apparent Ki = 0.05 microM), converted the EPR signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal designated MCR-BrPrSO3. 3-Fluoropropanesulfonate (apparent Ki < 50 microM) and 3-iodopropanesulfonate (apparent Ki < 1 microM) induced a signal identical to that induced by BrPrSO3 without affecting the line shape, despite the fact that the fluorine, bromine and iodine isotopes employed have nuclear spins of I = 1/2, I = 3/2 and I = 5/2, respectively. This finding suggests that MCR-BrPrSO3 is not the result of a close halogen-Ni(I) interaction. 7-Bromoheptanoylthreonine phosphate (BrHpoThrP) (apparent Ki = 5 microM), which is an inhibitory substrate analogue of 7-mercaptoheptanoylthreonine phosphate, converted the signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal, MCR-BrHpoThrP, similar but not identical to MCR-BrPrSO3. The results indicate that inhibition of MCR by the halogenated substrate analogues investigated above is not via oxidation of Ni(I)F430. The different MCR EPR signals are assigned to different enzyme/substrate and enzyme/inhibitor complexes.

Alkanesulfonates↗
PubMed1992

A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei.

We have recently reported that the thermophilic archaeon Methanobacterium wolfei contains two formylmethanofuran dehydrogenases, I and II. Formylmethanofuran dehydrogenase II, which is preferentially expressed in tungsten-grown cells, has been purified and shown to be a tungsten-iron-sulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum-grown cells and shown that it is a molybdenum-iron-sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per mol 146-kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non-heme iron but no tungsten (< 0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 U/mg) in that it catalyzed the oxidation of N-furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent Km for the electron acceptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N-terminal amino acid sequences determined for the alpha and beta subunits were identical up to residue 23, indicating that the two proteins are isoenzymes. The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.

Aldehyde Oxidoreductases↗
PubMed1992

H2-forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron-sulfur clusters in methanogenic archaea.

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2 = H4MPT) to methenyltetrahydromethanopterin (CH identical to H4MPT+) and H2 and was therefore named H2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H2 or CH2 = H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (Vmax) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between 3H2 and 1H+; (d) the enzyme catalyzed the reduction of CH identical to H4MPT+ with 3H2 generating [methylene-3H]CH2 = H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxin-type iron-sulfur protein. Properties of the H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.

Amino Acid Sequence↗
PubMed1992

Properties of the tungsten-substituted molybdenum formylmethanofuran dehydrogenase from Methanobacterium wolfei.

In Methanobacterium wolfei two formylmethanofuran dehydrogenases are present, one of which is a molybdenum- and the other a tungsten enzyme. We report here that also the 'molybdenum' enzyme contained tungsten when the archaeon was grown on molybdenum-deprived medium supplemented with tungstate (1 microM). Unexpectedly the tungsten-substituted molybdenum enzyme was catalytically active and displayed a rhombic EPR signal which was attributed to tungsten by the characteristic 183W splitting.

Aldehyde Oxidoreductases↗
PubMed1992

A tungsten-containing active formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei.

Methanobacterium wolfei is a thermophilic methanogenic archaeon which requires tungsten or molybdenum for growth. We have found that the organism contains two formylmethanofuran dehydrogenases, one of which is a tungsten enzyme. Indirect evidence indicates that the other formylmethanofuran dehydrogenase is a molybdenum enzyme. The tungsten enzyme was purified and characterized. The native enzyme had an apparent molecular mass of 130 kDa. SDS/PAGE revealed a composition of three subunits of apparent molecular mass 35, 51 and 64 kDa, the N-terminal amino acid sequences of two of which were determined. 0.3-0.4 mol tungsten/mol enzyme was found but no molybdenum. The pterin cofactor was identified as molybdopterin guanine dinucleotide. The purified enzyme exhibited a specific activity of 8.3 mumol.min-1.mg protein-1 and an apparent Km for formylmethanofuran and methylviologen of 13 microM and 0.4 mM, respectively. The optimum temperature for activity was 65 degrees C. At 40-60 degrees C, the rate increased with a Q10 of 1.9; the activation energy of the reaction was 45 kJ/mol. The enzyme was found to require potassium ions for thermostability. The oxygen-sensitive enzyme was not inactivated by cyanide.

Aldehyde Oxidoreductases↗
PubMed1992

Differential expression of the two methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum as determined immunochemically via isoenzyme-specific antisera.

Methanobacterium thermoautotrophicum contains two isoenzymes of methyl-coenzyme M reductase (MCR), MCR I and MCR II, which catalyze the methane-forming step and which together represent more than 10% of the cellular protein. We describe here the preparation of isoenzyme-specific antisera against the two MCR isoenzymes and their use in the quantitative immunochemical determination of the two isoenzymes in the methanogen. The relative and absolute cellular concentration of the two proteins is shown to be strongly affected by growth conditions such as the temperature, pH, and substrate concentration. Conditions were found yielding cells which contained essentially only MCR I or MCR II. Using antisera against MCR I and MCR II, MCR from other methanogens were immunochemically compared. Evidence is presented that Methanobacterium wolfei also contains two isoenzymes of MCR.

Immune Sera↗
PubMed1992

Isolation and characterization of polyferredoxin from Methanobacterium thermoautotrophicum. The mvhB gene product of the methylviologen-reducing hydrogenase operon.

The methylviologen-reducing hydrogenase operon of Methanobacterium thermoautotrophicum contains an open reading frame, mvhB, the product of which was predicted to have a molecular weight of 44 kDa and to contain as many as 48 iron atoms in 12 [4Fe-4S] clusters, and was therefore suggested to be a polyferredoxin. We have now, for the first time, isolated this polyferredoxin. Its identity with the mvhB gene product was evidenced by a comparison of the N-terminal amino acid sequence. The dark-brown protein of apparent molecular weight 44 kDa was found to contain 53 mol Fe and 43 mol acid-labile sulfur per mol. The UV/visible spectrum showed two maxima at 280 nm and 390 nm, and a shoulder at 308 nm. The A390/A280 ratio was 0.73. The molar extinction coefficient at 390 nm was 170,000 M-1.cm-1. In the dithionite reduced state the protein displayed an EPR spectrum like that of [4Fe-4S] clusters. The results indicate that the mvhB gene product is indeed a polyferredoxin.

Amino Acid Sequence↗
PubMed1992

N5-methyltetrahydromethanopterin: coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein.

An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N5-methyltetrahydromethanopterin (CH3-H4MPT) to coenzyme M (H-S-CoM) in methanogenic archaebacteria. With this method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri and in H2/CO(2)-grown Methanobacterium thermoautotrophicum were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH3-S-CoM formation from CH3-H4MPT (apparent Km = 50 microM) and H-S-CoM (apparent Km = 250 microM) of approximately 0.6 mumol.min-1.mg protein-1. For activity the presence of Ti(III) citrate (apparent Km = 15 microM) and of ATP (apparent Km = 30 microM) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH3-H4MPT in the absence of H-S-CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH3-H4MPT to H-S-CoM in the absence of ATP and Ti(III). Demethylation in the presence of H-S-CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.

Euryarchaeota↗
PubMed1991

Hydrogen-forming and coenzyme-F420-reducing methylene tetrahydromethanopterin dehydrogenase are genetically distinct enzymes in Methanobacterium thermoautotrophicum (Marburg).

A coenzyme-F420-reducing and an H2-forming methylenetetrahydromethanopterin dehydrogenase have been isolated from Methanobacterium thermoautotrophicum (Marburg). Indirect evidence suggested that the former enzyme (32 kDa) might be derived from the latter enzyme (42 kDa) by proteolysis. To test this hypothesis the gene sequence of the H2-forming dehydrogenase was determined and compared with the N-terminal amino acid sequence of the F420-reducing dehydrogenase. No corresponding sequences were found indicating that the two dehydrogenases are genetically distinct enzymes. With purified enzyme preparations it is shown that the activity of the F420-reducing dehydrogenase is inhibited in the presence of the H2-forming enzyme. This finding is discussed in terms of substrate competition.

Amino Acid Sequence↗
PubMed1991

Methyl-coenzyme M reductase preparations with high specific activity from H2-preincubated cells of Methanobacterium thermoautotrophicum.

The study of the nickel enzyme methyl-coenzyme M reductase from methanogenic bacteria has been hampered until now by the fact that upon cell rupture the activity of the enzyme always dropped to at best only a few percent of its in vivo activity. We describe here that when Methanobacterium thermoautotrophicum cells were preincubated with 100% H2 before disintegration methyl-coenzyme M reductase activity stayed high. The cell extracts with a specific activity of 2 U/mg protein exhibited two nickel-derived EPR signals, designated MCR-red1 and MCR-red2, previously only observed in intact cells. The enzyme was purified 10-fold to a specific activity of 20 U/mg in the presence of methyl-coenzyme M, which stabilized both the activity and the EPR signal MCR-red1. The enzyme preparation displayed an UV/Vis spectrum with an absorption maximum at 386 nm and a shoulder at 420 nm. Upon inactivation of the enzyme with O2 or CHCl3, the maximum at 386 nm and the EPR signals MCR-red1 and MCR-red2 disappeared.

Electron Spin Resonance Spectroscopy↗
PubMed1991

N5, N10-methylenetetrahydromethanopterin dehydrogenase (H2-forming) from the extreme thermophile Methanopyrus kandleri.

Methanopyrus kandleri is a novel abyssal methanogenic archaebacterium growing at 110 degrees C on H2 and CO2. The N5, N10-methylenetetrahydromethanopterin dehydrogenase, an enzyme involved in methanogenesis from CO2 and H2, was purified from this hyperthermophile and characterized. The dehydrogenase was found to be composed of only one polypeptide of apparent molecular mass 44 kDa. The UV/Vis spectrum was similar to that of albumin. The protein catalyzed the reversible dehydrogenation of N5, N10-methylenetetrahydromethanopterin (CH2 = H4MPT) to N5, N10-methenyltetrahydromethanopterin (CH identical to H4MPT+) and molecular hydrogen: CH2 = H4MPT H+ in equilibrium CH identical to H4MPT+ +H2. The rate of CH2 = H4MPT dehydrogenation (apparent Vmax) at 65 degrees C and pH 5.8 was 1500 U/mg, the apparent Km for CH2 = H4MPT was 50 microM, the Arrhenius activation energy was 52 kJ/mol, and the Q10 between 30 degrees C and 70 degrees C was 2.0. The specific activity increased hyperbolically with the proton concentration between pH 7 and pH 4.5. The purified dehydrogenase did not catalyze the reduction of viologen dyes, of coenzyme F420, and of pyridine nucleotides with either CH2 = H4MPT or H2. For activity the CH2 = H4MPT dehydrogenase required the presence of salts. Fifty percent of maximal activity was reached at salt concentrations of 100 mM, potassium phosphate, potassium chloride, and sodium chloride being almost equally effective in stimulating the enzyme activity. Cell extracts of M. kandleri did not loose CH2 = H4MPT dehydrogenase activity when incubated at 90 degrees C for 60 min. The purified enzyme, however, proved very thermolabile. The purified enzyme, however, proved very thermolabile.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri.

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110 degrees 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 alpha-, beta- and gamma-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 gamma-subunit was determined. A comparison with the N-terminal sequences of the gamma-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 degrees C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran:tetrahydro-methanopterin formyltransferase, 13 U/mg; N5,N10-methylenetetrahydromethanopterin cyclohydrolase, 14U/mg; N5,N10-methenyltetrahydromethanopterin dehydrogenase (H2-forming), 33 U/mg; N5,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-gamma-glutamyl residues.

Amino Acid Sequence↗
PubMed1990

Single step purification of methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum by specific binding to blue sepharose CL-6B.

Methylenetetrahydromethanopterin reductase from metanogenic archaebacteria catalyzes the reversible reduction of N5,N10-methylenetetrahydromethanopterin to N5-methyltetrahydromethanopterin with reduced coenzyme F420 as electron donor. The enzyme is involved in methane formation from CO2 and in methanol disproportionation to CO2 and CH4. We report here that the reductase from Methanobacterium thermoautotrophicum specifically binds to Blue Sepharose CL-6B. Binding was competitive with coenzyme F420 rather than with NAD, NADP, FAD, FMN, AMP, ADP and ATP. The reductase could also be desorbed with salt. Based on this property an affinity chromatographic procedure for the purification of the enzyme was developed.

Binding, Competitive↗
PubMed1979

Ferredoxin degradation in growing Clostridium pasteurianum during periods of iron deprivation.

Clostridium pasteurianum was grown in batch cultures on media with an initial iron concentration of 10 micron. The uptake of iron and the synthesis of ferredoxin was followed. All the iron present in the medium was taken up by the cells before 50% of the final cell density was attained. The bacteria then continued to grow in the complete absence of exogenous iron. Ferrodoxin was synthesized during growth until the exogenous iron concentration dropped below 1 micron. During growth in the absence of iron ferredoxin was degraded with the result that at the end of growth the cells did not contain ferredoxin. The specific activity of the iron sulfur protein, pyruvate synthase (E.C. 1.2.7.1), remained constant during growth of C. pasteurianum in the absence of exogenous iron. This finding suggest that ferredoxin was used as an endogenous source of iron for the synthesis of essential iron proteins during periods of iron deprivation.

Biodegradation, Environmental↗
PubMed1979

Nickel, cobalt, and molybdenum requirement for growth of Methanobacterium thermoautotrophicum.

Growth of Methanobacterium thermoautotrophicum on H2 and CO2 as sole energy and carbon sources was found to be dependent on Ni, Co, and Mo. At low concentrations of Ni (less than 100 nM), Co (less than 10 nM) and Mo (less than 10 nM) the amount of cells formed was roughly proportional to the amount of transition metal added to the medium; for the formation of 1 g cells (dry weight) approximately 150 nmol NiCl2, 20 nmol CoCl2 and 20 nmol Na2MoO4 were required. A dependence of growth on Cu, Mn, Zn, Ca, Al, and B could not be demonstrated. Conditions are described under which the bacterium grew exponentially with a doubling time of 1.8 h up to a cell density of 2 g cells (dry weight)/l.

Carbon Dioxide↗