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PubMed2006

A mathematical model for the kinetics of Methanobacterium bryantii M.o.H. considering hydrogen thresholds.

We develop a kinetic model that builds on the foundation of classic Monod kinetics, but incorporates new phenomena such as substrate thresholds and survival mode observed in experiments with the H2-oxidizing methanogen Methanobacterium bryantii M.o.H. We apply our model to the experimental data presented in our companion paper on H2 thresholds. The model accurately describes H2 consumption, CH4 generation, biomass growth, substrate thresholds, and survival state during batch experiments. Methane formation stops when its Gibbs free energy is equal zero, although this does not interrupt H2 oxidation. The thermodynamic threshold for H2 oxidation occurs when the free energy for oxidizing H2 and transferring electrons to biomass is no longer negative, at approximately 0.4 nM. This threshold is not controlled by the Gibbs free energy equation of methanogenesis from H2 + HCO3- as we show in our companion paper. Beyond this threshold, the microorganisms shift to a low-maintenance metabolism called "the survival state" in response to extended H2 starvation; adding the starvation response as another new feature of the kinetic model. A kinetic threshold (or S (min)), a natural feature of the Monod kinetics, is also captured by the model at H2 concentration of around approximately 2,400 nM. S (min) is the minimum substrate concentration to maintain steady-state biomass concentration. Our model will be useful for interpreting threshold results and designing new studies to understand thresholds and their ecological implications.

Computer Simulation↗
PubMed1994

Acylation of proteins of the archaebacteria Halobacterium cutirubrum and Methanobacterium thermoautotrophicum.

Although the membrane lipids of extremely halophilic archaebacteria are exclusively derived from diphytanylglycerol diether, which is non-acylated, small amounts of fatty acids have been detected in these organisms. These fatty acids are formed by the action of a fatty acid synthase (FAS), shown to be present in the extreme halophile Halobacterium cutirubrum, despite the fact that only a fraction of the activity of FAS remains at the high salt concentration (> 4 M) present in the cytoplasm. It has now been demonstrated that fatty acids do not occur in lipid-bound form but largely in the form of acylated proteins in the red membrane of H. cutirubrum. In contrast, the bacteriorhodopsin of the purple membrane of this extreme halophile does not appear to be acylated. The thermophilic methanogen, Methanobacterium thermoautotrophicum had a much higher fatty acid synthase activity than the extreme halophile, and the synthase activity of the methanogen was optimal under its normal (anaerobic) growth conditions. The methanogen also utilized the resulting fatty acids to acylate its membrane proteins. The major fatty acids in both organisms were palmitic and stearic acids with small amounts of myristic and 18:1 acids, and these were bound to protein through both ester and amide linkages.

Acylation↗
PubMed1992

A possible new class of ribonucleotide reductase from Methanobacterium thermoautotrophicum.

The ribonucleotide reductase from the strictly anaerobic methanogen Methanobacterium thermoautotrophicum has been partially purified by ion-exchange and gel-filtration chromatography. Its molecular weight is estimated to be 100,000 by the latter step. Unlike all previously studied ribonucleotide reductases, the enzyme does not employ dithiol compounds such as dithiothreitol as artificial electron donors in in vitro assays. Inhibition of the enzyme by S-adenosylmethionine, oxygen, and azide further distinguishes it from the Escherichia coli anaerobic enzyme, the iron- and manganese-containing, and the adenosylcobalamin-dependent enzymes. Our preliminary results suggest that this enzyme has an activation mechanism different from the known classes of ribonucleotide reductases.

Adenosine↗
PubMed1991

Molybdopterin adenine dinucleotide and molybdopterin hypoxanthine dinucleotide in formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum (Marburg).

Formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum was purified to apparent homogeneity and found to contain per mol (apparent molecular mass 110 kDa) 0.6 mol molybdenum, 4 mol non-heme iron, 4 mol acid-labile sulfur, and in addition, 0.7 mol of a pterin-containing co-factor (apparent molecular mass 800 Da) which has been characterized. The pterin material was extracted after alkylation by iodoacetamide and the extract subjected to HPLC on Lichrospher 100 RP-18. Three pterin compounds were resolved. On the basis of their UV/visible spectra and of the products formed after cleavage by nucleotide pyrophosphatase and alkaline phosphatase they were identified as the [di(carboxamidomethyl)]-derivatives of molybdopterin guanine dinucleotide (MGD), of molybdopterin adenine dinucleotide (MAD), and of molybdopterin hypoxanthine dinucleotide (MHD). The three pterin dinucleotides were present in the proportions 1:0.4:0.1.

Adenine Nucleotides↗
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↗
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

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↗
PubMed1996

Identification of the gltX gene encoding glutamyl-tRNA synthetase from Methanobacterium thermoautotrophicum.

The gltX gene encoding glutamyl-tRNA synthetase from Methanobacterium thermoautotrophicum has been cloned, sequenced, and identified. The gene is located immediately downstream of idsA in an operon containing at least three additional ORFs. The deduced protein sequence from gltX contains conserved regions (HIGH and KMSKS) indicative of a class I aminoacyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1992

Isolation of a 5-hydroxybenzimidazolyl cobamide-containing enzyme involved in the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in Methanobacterium thermoautotrophicum.

Formaldehyde conversion into methyl-coenzyme M involves (a) reaction of the substrate with 5,6,7,8-tetrahydromethanopterin (H4MPT) giving 5,10-methylene-H4MPT, followed by its reduction to 5-methyl-H4MPT and (b) transfer of the methyl group from the latter compound to coenzyme M. The reactions were studied in a resolved system from Methanobacterium thermoautotrophicum strain delta H. The first part (a) of the reactions was catalyzed by the 55% ammonium sulfate supernatant of cell-free extracts. The methyltransferase step (b) was dependent on an oxygen-sensitive enzyme, called methyltransferase a (MTa). Isolation of MTa was achieved by gel filtration on Sephacryl S-400. MTa was a high-molecular-weight complex of at least 2000 kDa and between 900 to 1500 kDa when purified in the absence and presence of the detergent CHAPS, respectively. The enzyme consisted of 100 kDa units composed of three subunits in an alpha beta gamma configuration with apparent molecular masses of 35, 33 and 31 kDa, respectively. The corrinoid, 5-hydroxybenzymidazolyl cobamide (B12HBI, Factor III) copurified with MTa and the latter contained 2 nmol B12HBI per mg protein. B12HBI present in MTa could be methylated under the appropriate conditions by 5-methyl-H4MPT. These findings suggest that the corrinoid is a prosthetic group of MTa. MTa may be homologous to the corrinoid membrane protein purified before from M. thermoautotrophicum strain Marburg (Schulz, H., Albracht, S.P.J., Coremans, J.M.C.C. and Fuchs, G. (1988) Eur. J. Biochem. 171, 589-597).

Cobamides↗
PubMed1994

Purification and characterization of inorganic pyrophosphatase from Methanobacterium thermoautotrophicum (strain delta H).

Inorganic pyrophosphatase (EC 3.6.1.1.) has been isolated from the archaebacterium Methanobacterium thermoautotrophicum (strain delta H). The enzyme was purified 850-fold in three steps to electrophoretic homogeneity. The soluble pyrophosphatase consists of four identical subunits: the molecular mass of the native enzyme estimated by gel filtration was approx. 100 kDa and denaturing polyacrylamide gel electrophoresis gave a single band of 25 kDa. The enzyme also may occur as an active dimer formed by dissociation of the tetramer. The pyrophosphate showed an optimal activity at 70 degrees C and a pH of 7.7 (at 60 degrees C) and was not influenced by dithiothreitol, sodium dithionite or potassium chloride. The enzyme was very specific for pyrophosphate (PPi) and Mg2+. Magnesium could be partially replaced by Co2+ (15%). The reaction was inhibited for 60% by 1 mM Mn2+ in the presence of 24 mM Mg2+. In addition, the enzyme was inhibited by potassium fluoride (50% at 0.9 mM). Kinetic analysis revealed positive co-operativity for both Mg2+ and PPi with Hill coefficients of 3.3 and 2.0, respectively. Under the experimental conditions at which the enzyme was present as its dimer, the apparent Km of PPi and magnesium were determined and were approx. 0.16 mM and 4.9 mM, respectively; Vmax was estimated at about 570 U/mg.

Enzyme Stability↗
PubMed1994

Characterization and partial purification of an ATPase and inorganic pyrophosphatase of the archaebacterium Methanobacterium thermoautotrophicum.

ATPase and inorganic pyrophosphatase (PPase) activities have been detected in several methanogenic bacteria. These activities are believed to play a crucial role in energy metabolism. In the present study we have investigated some characteristics of the ATPase and the inorganic PPase activities of Methanobacterium thermoautotrophicum. Although these proteins migrate identically on non-dissociating gels, they are catalyzed by distinct enzymes which are separable by biochemical purification methods. The partially purified enzymes are composed of at least two subunits. The ATPase subunits have molecular masses of about 43 and 33 kDa and the inorganic PPase such of about 31 and 25 kDa. After purification, the PPase and the ATPase did not hydrolyze ATP and PP(i), respectively. The membrane-bound ATPase and PPase activities are distinguished in response to sodium fluoride, by the effects of divalent cations, by the temperature ranges for activities and the solubilization behaviour by different extractants. Most investigated catalytic and structural properties of the ATPase do not suit the current criteria for classifying this enzyme under either the F-, V- or P-ATPases.

Adenosine Triphosphatases↗
PubMed1996

The hemA gene encoding glutamyl-tRNA reductase from the archaeon Methanobacterium thermoautotrophicum strain Marburg.

In archaea the first general tetrapyrrole precursor 5-aminolevulinic acid (ALA) is formed via the tRNA-dependent five-carbon pathway from glutamate. We have cloned the hemA gene encoding the central enzyme of the pathway glutamyl-tRNA reductase from the methanogenic archaeon Methanobacterium thermoautotrophicum by complementation of an Escherichia coli hemA mutant to ALA prototrophy. An 1194 bp open reading frame that encodes a 398 amino acid polypeptide with the calculated M, 44,509 was detected. The deduced amino acid sequence showed 20-35% amino acid identity to bacterial HemAs with the highest identity score to the Pseudomonas aeruginosa HemA. An identity of approximately 22% was found to plant HemAs. Glutamyl-tRNA reductase activity was shown for the M. thermoautotrophicum HemA after overexpression in E. coli and partial purification. The enzymatic reaction catalyzed by the partially purified enzyme revealed a temperature optimum of 65 degrees C at an optimal pH of 7.0. The reductase utilized preferentially NADPH for the reduction of the activated carboxyl group. The presence of ATP and GTP showed no obvious influence on catalysis.

Aldehyde Oxidoreductases↗
PubMed2004

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions.

The first inhibition study of the beta-class carbonic anhydrase (CA, EC 4.2.1.1) from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions is reported here. Inhibition data of the alpha-class human isozymes hCA I and hCA II (cytosolic) as well as the membrane-bound isozyme hCA IV and the gamma-class enzyme from another archaeon, Methanosarcina thermophila (Cam) with a large number of anionic species such as halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, bisulfite, sulfate, etc., are also provided for comparison. The best Cab anion inhibitors were thiocyanate and hydrogen sulfide, with inhibition constants in the range of 0.52-0.70 mM, whereas cyanate, iodide, carbonate, and nitrate were weaker inhibitors (Ki's in the range of 7.8-13.2 mM). Fluoride, chloride, and sulfate do not inhibit this enzyme appreciably, whereas the CA substrate bicarbonate, or other anions, such as bromide, nitrite, bisulfite, or sulfamate behave as weak inhibitors (Ki in the range of 40-45 mM). It is interesting to note that the metal poison, coordinating anions cyanide and azide are also rather weak Cab inhibitors (Ki in the range of 27-55 mM), whereas sulfamide is a very weak Cab inhibitor (Ki of 103 mM), although it strongly inhibits Cam (Ki of 70 microM). Surprisingly, phenylboronic and phenylarsonic acids, which have been investigated for the inhibition of all these CAs for the first time, showed very weak activity against the alpha-CA isozymes, but were effective Cab and Cam inhibitors. The best Cab inhibitors were just these two compounds (Ki's of 0.20-0.33 mM), whereas the best Cam inhibitor was sulfamic acid (Ki of 96 nM). These major differences of behavior between the diverse CAs investigated here toward anion inhibitors can be difficultly explained considering the convergent evolution of so diverse enzymes for the binding and turnover of small molecules such as carbon dioxide and anions.

Anions↗
PubMed1996

The presence of H+ and Na+ -linked Ca2+ extruding systems in Methanobacterium thermoautotrophicum.

The effects of monovalent cations (Na+, K+ and choline+) and the uncoupler 3,3',4',5-tetrachlorosalicylanilide (TCS) were tested on 45Ca2+ uptake by non-energized cells of Methanobacterium thermoautotrophicum. 45Ca2+ uptake was stimulated by the addition of K+ and (less) by choline+ while Na+ slowed down and even reversed it, thereby mimicking the energization of cells. The uncoupler agent, TCS, suppressed 45Ca2+ uptake in non-energized cells in the presence or absence of Na+ but in cells energized in an atmosphere of CO2+H2 it exerted a stimulating effect. Uncoupled 45Ca2+ efflux was measured in cells pre-loaded with 45Ca2+ by means of the divalent ionophore A23187 following its washing out by buffer containing serum albumin. The efflux was temperature-dependent and was stimulated by external 40Ca2+ and Na+. In the absence of Na+, the uncoupled efflux was completely inhibited by TCS, whereas in the presence of Na+, TCS was without any effect. The results are in agreement with the model in which the Ca2+ influx pathway is represented by a membrane potential-driven uniport whereas Ca2+ efflux is mediated by two transport systems - Na+/Ca2+ and H+/Ca2+ antiporters - whose participation in the total efflux is dependent on the energy of the corresponding gradients of driving ions.

Calcium↗
PubMed1997

Medium-reductant directed expression of methyl coenzyme M reductase isoenzymes in Methanobacterium thermoautotrophicum (strain deltaH).

Methanobacterium thermoautotrophicum was grown in a chemostat under various controlled conditions in the presence of either sodium sulfide or sodium thiosulfate. After establishment of the steady state, cells were taken and examined for expression of the mRNA transcripts coding for the different forms of methyl coenzyme M reductase (MCR) and methylene tetrahydomethanopterin dehydrogenase (MDH). MCR isoenzyme II expression varied most markedly. Expression was found not only to depend on known parameters temperature, pH and gassing rate, but also on the medium composition, especially the reductant present.

Base Sequence↗
PubMed1997

Isolation and characterization of a neomycin-resistant mutant of Methanobacterium thermoautotrophicum with a lesion in Na+-translocating ATPase (synthase).

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na+-translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three-fold as compared with the wild-type strain. In contrast to wild-type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na+ ions. The methane formation orate of the mutant cells at pH 7.5 under non-growing conditions was nearly twice that of the wild-type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild-type under the same conditions, and contrary to the wild-type was not stimulated by Na+ ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild-type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (-223 mV and -230 mV respectively). The Mg2+-dependent ATPase activity of the 10(5) x g supernatant of broken cells from the mutant cells was 30% higher than in the wild-type. On the other hand, two bands with Mg2+-dependent ATPase activity were identified by native PAGE in this fraction in both wild-type as well as in mutant. These data suggest that the binding of Na+-translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.

Adenosine Triphosphatases↗
PubMed1998

His84 rather than His35 is the active site histidine in the corrinoid protein MrtA of the energy conserving methyltransferase complex from Methanobacterium thermoautotrophicum.

The energy conserving corrinoid containing MtrA-H complex from Methanobacterium thermoautotrophicum is composed of eight different subunits of which MtrA harbors the corrinoid prosthetic group, the corrinoid being bound in the base-off/His-on configuration. Based on sequence comparisons it was recently proposed that His35 of MtrA is the active site histidine. We report here that His84 rather than His35 is the axial ligand to the cobamide in MtrA.

Amino Acid Sequence↗
PubMed1998

F420H2:NADP oxidoreductase from Methanobacterium thermoautotrophicum: identification of the encoding gene via functional overexpression in Escherichia coli.

F420H2:NADP oxidoreductase is found in methanogenic, sulfate-reducing and halophilic archaea and also in some bacteria. The putative gene encoding the enzyme was cloned from Methanobacterium thermoautotrophicum (strain Marburg) and heterologously expressed in Escherichia coli. The overproduced active enzyme was purified, characterized and crystallized.

Chromatography↗