Biomedical subjects

R K Thauer

Publications and source records attributed to R K Thauer.

At least 55 records · Page 3Linked to original sources

PubMed1998

Two malate dehydrogenases in Methanobacterium thermoautotrophicum.

Methanobacterium thermoautotrophicum (strain Marburg) was found to contain two malate dehydrogenases, which were partially purified and characterized. One was specific for NAD+ and catalyzed the dehydrogenation of malate at approximately one-third of the rate of oxalacetate reduction, and the other could equally well use NAD+ and NADP+ as coenzyme and catalyzed essentially only the reduction of oxalacetate. Via the N-terminal amino acid sequences, the encoding genes were identified in the genome of M. thermoautotrophicum (strain DeltaH). Comparison of the deduced amino acid sequences revealed that the two malate dehydrogenases are phylogenetically only distantly related. The NAD+-specific malate dehydrogenase showed high sequence similarity to L-malate dehydrogenase from Methanothermus fervidus, and the NAD(P)+-using malate dehyrogenase showed high sequence similarity to L-lactate dehydrogenase from Thermotoga maritima and L-malate dehydrogenase from Bacillus subtilis. A function of the two malate dehydrogenases in NADPH:NAD+ transhydrogenation is discussed.

Acetates↗

PubMed1998

The formylmethanofuran dehydrogenase isoenzymes in Methanobacterium wolfei and Methanobacterium thermoautotrophicum: induction of the molybdenum isoenzyme by molybdate and constitutive synthesis of the tungsten isoenzyme.

Formylmethanofuran dehydrogenase catalyzes the first step in methane formation from CO2 in methanogenic archaea. Methanobacterium wolfei and Methanobacterium thermoautotrophicum have been shown to contain two isoenzymes, a tungsten-containing isoenzyme (Fwd) and a molybdenum-containing isoenzyme (Fmd). We report here that in both thermophilic organisms the encoding genes are organized in a highly conserved fwdHFGDACB tungsten operon and in an fmdECB molybdenum operon. In both organisms, the tungsten isoenzyme was found to be constitutively transcribed, whereas the transcription of the molybdenum operon was induced by molybdate. Induction by molybdate was not significantly affected by tungstate.

Aldehyde Oxidoreductases↗

PubMed1998

Activation and thermostabilization effects of cyclic 2, 3-diphosphoglycerate on enzymes from the hyperthermophilic Methanopyrus kandleri.

Enzymes involved in methane formation from carbon dioxide and dihydrogen in Methanopyrus kandleri require high concentrations (> 1 M) of lyotropic salts such as K2HPO4/KH2PO4 or (NH4)2SO4 for activity and for thermostability. The requirement correlates with high intracellular concentrations of cyclic 2,3-diphosphoglycerate (cDPG; approximately 1 M) in this hyperthermophilic organism. We report here on the effects of potassium cDPG on the activity and thermostability of the two methanogenic enzymes cyclohydrolase and formyltransferase and show that at cDPG concentrations prevailing in the cells the investigated enzymes are highly active and completely thermostable. At molar concentrations also the potassium salts of phosphate and of 2,3-bisphosphoglycerate, the biosynthetic precursor of cDPG, were found to confer activity and thermostability to the enzymes. Thermodynamic arguments are discussed as to why cDPG, rather than these salts, is present in high concentrations in the cells of Mp. kandleri.

2,3-Diphosphoglycerate↗

PubMed1998

Overproduction and one-step purification of the N5,N10-methenyltetrahydromethanopterin cyclohydrolase (Mch) from the hyperthermophilic Methanopyrus kandleri.

N5,N10-Methenyltetrahydromethanopterin cyclohydrolase (Mch) is an enzyme involved in methanogenesis from CO2 and H2 which represents the energy metabolism of Methanopyrus kandleri, a methanogenic Archaeon growing at a temperature optimum of 98 degrees C. The gene mch from M. kandleri was cloned, sequenced, and expressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% in one step by chromatography on phenyl Sepharose in 80% ammonium sulfate. From 3.5 g cells (250 mg protein), approximately 18 mg cyclohydrolase was obtained. The purified enzyme showed essentially the same catalytic properties as the enzyme purified from M. kandleri cells. The primary structure and properties of the cyclohydrolase are compared with those of the enzyme from Methanococcus jannaschii (growth temperature optimum 85 degrees C), from Methanobacterium thermoautotrophicum (65 degrees C), and from Methanosarcina barkeri (37 degrees C). Of the four enzymes, that from M. kandleri has the lowest isoelectric point (3.8) and the lowest hydrophobicity of amino acid composition. Besides, it has the highest relative content of glutamate, leucine, and valine and the lowest relative content of isoleucine, serine, and lysine. Some of these properties are unusual for enzymes from hyperthermophilic organisms. They may reflect the observation that the cyclohydrolase from M. kandleri is not only adapted to hyperthermophilic conditions but also to the high intracellular concentrations of lyotrophic salts prevailing in this organism.

Amino Acid Sequence↗

PubMed1998

Active sites of transition-metal enzymes with a focus on nickel.

Since 1995, crystal structures have been determined for many transition-metal enzymes, in particular those containing the rarely used transition metals vanadium, molybdenum, tungsten, manganese, cobalt and nickel. Accordingly, our understanding of how an enzyme uses the unique properties of a specific transition metal has been substantially increased in the past few years. The different functions of nickel in catalysis are highlighted by describing the active sites of six nickel enzymes - methyl-coenyzme M reductase, urease, hydrogenase, superoxide dismutase, carbon monoxide dehydrogenase and acetyl-coenzyme A synthase.

Binding Sites↗

PubMed1998

The NADP-dependent methylene tetrahydromethanopterin dehydrogenase in Methylobacterium extorquens AM1.

An NADP-dependent methylene tetrahydromethanopterin (H4MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO2 in Methylobacterium extorquens AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be the mtdA gene product. The purified enzyme catalyzed the dehydrogenation of methylene H4MPT with NADP+ rather than with NAD+, with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H4F) with NADP+. With methylene H4F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (Vmax/Km) were approximately 20-fold lower than with methylene H4MPT. Whereas the dehydrogenation of methylene H4MPT (E0 = -390 mV) with NADP+ (E0 = -320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H4F (E0 = -300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from M. extorquens AM1 with those of methylene H4F dehydrogenases from other bacteria and eucarya and with those of methylene H4MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).

Amino Acid Sequence↗

PubMed1997

Identification of the active site histidine in the corrinoid protein MtrA 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. EPR spectroscopic evidence has recently been presented for a histidine residue as a cobalt ligand of the cobamide [Harms, U. & Thauer, R. K. (1996a) Eur. J. Biochem. 241, 149-154]. This active site histidine was now identified by site-directed mutagenesis to be His84 in the MtrA sequence that contains three histidines. This result was substantiated by sequence comparison of MtrA from M. thermoautotrophicum, Methanococcus jannaschii, and Methanopyrus kandleri and of MtxA from Methanosarcina harkeri showing that only His84 is conserved. For comparison, the DNA sequences of the mtrEDCBAGH operon in M. kandleri and of the mtxXAH operon in M. barkeri were determined.

Amino Acid Sequence↗

PubMed1997

Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation.

Methyl-coenzyme M reductase (MCR), the enzyme responsible for the microbial formation of methane, is a 300-kilodalton protein organized as a hexamer in an alpha2beta2gamma2 arrangement. The crystal structure of the enzyme from Methanobacterium thermoautotrophicum, determined at 1.45 angstrom resolution for the inactive enzyme state MCRox1-silent, reveals that two molecules of the nickel porphinoid coenzyme F430 are embedded between the subunits alpha, alpha', beta, and gamma and alpha', alpha, beta', and gamma', forming two identical active sites. Each site is accessible for the substrate methyl-coenzyme M through a narrow channel locked after binding of the second substrate coenzyme B. Together with a second structurally characterized enzyme state (MCRsilent) containing the heterodisulfide of coenzymes M and B, a reaction mechanism is proposed that uses a radical intermediate and a nickel organic compound.

Binding Sites↗

PubMed1997

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri. Zinc dependence and thermodynamics of the methanol:cob(I)alamin methyltransferase reaction.

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl-coenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated methyltransferases 1 (MT1) and 2 (MT2). Transferase MT1, which is composed of a 50-kDa subunit, MtaB, and a 27-kDa corrinoid-harbouring subunit, MtaC, has been shown recently to catalyze the methylation of free cob(I)alamin with methanol [Sauer, K., Harms, U. & Thauer, R. K. (1997) Eur. J. Biochem. 243, 670-677]. We report here that this reaction is catalyzed by subunit MtaB overproduced in Escherichia coli. MtaB also catalyzed the formation of methanol from methylcobalamin and H2O, the hydrolysis being associated with a free-energy change deltaG(o)' of approximately +7.0 kJ/mol. MtaB was found to contain 1 mol zinc, and its activity to be zinc dependent (pK(Zn2+) = 9.3). The zinc dependence of the MT2 (MtaA)-catalyzed reaction is also described (pK(Zn2+) = 9.6).

Archaeal Proteins↗

PubMed1997

The active species of 'CO2' utilized by formylmethanofuran dehydrogenase from methanogenic Archaea.

Formylmethanofuran dehydrogenase from methanogenic Archaea catalyzes the reversible conversion of CO2 and methanofuran to formylmethanofuran, which is an intermediate in methanogenesis from CO2, a biological process yielding approximately 0.3 billion tons of CH4 per year. With the enzyme from Methanosarcina barkeri, it is shown that CO2 rather than HCO3- is the active species of 'CO2' utilized by the dehydrogenase. Evidence is also presented that the enzyme catalyzes a methanofuran-dependent exchange between CO2 and the formyl group of formylmethanofuran. The results are consistent with N-carboxymethanofuran being an intermediate in CO2 reduction to formylmethanofuran.

Aldehyde Oxidoreductases↗

PubMed1997

Overexpression of the coenzyme-F420-dependent N5,N10-methylenetetrahydromethanopterin dehydrogenase gene from the hyperthermophilic Methanopyrus kandleri.

The mtd gene encoding coenzyme-F420-dependent N5,N10-methylenetetrahydromethanopterin dehydrogenase (Mtd) in the hyperthermophilic Methanopyrus kandleri has been cloned, sequenced and functionally overexpressed in Escherichia coli. The overproduced enzyme was purified in a 90% yield to apparent homogeneity by means of only one chromatographic step. Its thermostability properties and most of its catalytic properties were the same as those of the native enzyme purified directly from M. kandleri. Only the dependence of the activity on the concentration of lyotropic salts differed slightly. Northern blot analysis revealed that in M. kandleri the mtd gene is monocistronically transcribed.

Amino Acid Sequence↗

PubMed1997

Structures and functions of four anabolic 2-oxoacid oxidoreductases in Methanobacterium thermoautotrophicum.

Methanobacterium thermoautotrophicum (strain Marburg), which grows autotrophically on H2 and CO2, was found to contain 2-oxoisovalerate oxidoreductase (Vor) and indolepyruvate oxidoreductase (Ior) besides pyruvate oxidoreductase (Por) and 2-oxoglutarate oxidoreductase (Kor). So far, Vor and Ior have only been detected in peptide-utilizing hyperthermophilic Archaea. The four 2-oxoacid oxidoreductases were purified and characterized with respect to their subunit composition, N-terminal amino acid sequences, and catalytic properties. Por and Kor were composed of four different subunits, Vor was composed of three different subunits, and Ior of two different subunits. Comparisons of the N-terminal amino acid sequences revealed that the four enzymes are structurally related to each other and to the respective enzymes from Pyrococcus and Thermococcus sp. Vor from M. thermoautotrophicum differed from Vor from Pyrococcus furiosus in being composed of only three instead of four different subunits. Evidence is presented that in the autotrophic methanogen the four 2-oxoacid oxidoreductases have anabolic functions, Vor and Ior being involved in the biosynthesis of amino acids from fatty acids taken up from the growth medium, as shown by 14C-labelling studies.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1997

Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri is not a flavoenzyme.

Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri (MbHdrDE) is a membrane-bound enzyme composed of a 46-kDa subunit MbHdrD and a 23-kDa subunit MbHdrE. The enzyme has been shown to contain 0.6 mol heme and 20 mol Fe/S per mol heterodimer. In addition, substoichiometric amounts of FAD, thought to be an essential component of the active enzyme, were detected. We have now obtained preparations of active heterodisulfide reductase in high yields completely devoid of a flavin. Cloning and sequencing of the genes encoding MbHdrD and MbHdrE, which were found to form a transcription unit hdrED, revealed that both subunits also lack an FAD-binding motif. MbHdr thus differs from heterodisulifde reductase from Methanobacterium thermoautotrophicum (MtHdr), which is a flavo iron-sulfur protein composed of the subunits MtHdrA (80 kDa), MtHdrB (36 kDa) and MtHdrC (21 kDa), the subunit HdrA harboring the flavin-binding site. Sequence comparisons revealed that the N-terminal third of MbHdrD, which contained two sequence motifs for [4Fe-4S] clusters, is similar to MtHdrC and that the C-terminal two thirds of MbHdrD are similar to MtHdrB. Thus, MbHdrD and MtHdrC are structurally equivalent subunits. MbHdrE shows sequence similarity to b-type cytochromes, in agreement with the finding that this subunit contains a heme. These and other results indicate that MbHdrD harbors the active site of heterodisulfide reduction and that a flavin is not involved in catalysis. Since MbHdrD contains only iron-sulfur clusters, a mechanism of disulfide reduction involving one electron rather than two electron-transfer reactions has to be considered such as operative in ferredoxin :thioredoxin reductases from chloroplasts and cyanobacteria.

Amino Acid Sequence↗

PubMed1997

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri. Purification, properties and encoding genes of the corrinoid protein MT1.

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methylcoenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated MT1 and MT2. Transferase MT1 is a corrinoid protein. The purification, catalytic properties and encoding genes of MT2 (MtaA) have been described previously [Harms, U. and Thauer, R.K. (1996) Eur. J. Biochem. 235, 653-659]. We report here on the corresponding analysis of MT1. The corrinoid protein MT1 was purified to apparent homogeneity and showed a specific activity of 750 mumol min-1 mg-1. The enzyme catalyzed the methylation of its bound corrinoid in the cob(I)amide oxidation state by methanol. In addition to this automethylation, the purified enzyme was found to catalyze the methylation of free cob(I)alamin to methylcob(III)alamin. It was composed of two different subunits designated MtaB and MtaC, with apparent molecular masses of 49 kDa and 24 kDa, respectively. The subunit MtaC was shown to harbour the corrinoid prosthetic group. The genes mtaB and mtaC were cloned and sequenced. They were found to be juxtapositioned and to form a transcription unit mtaCB. The corrinoid-harbouring subunit MtaC exhibits 35% sequence similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli.

Amino Acid Sequence↗

PubMed1997

Purified methyl-coenzyme-M reductase is activated when the enzyme-bound coenzyme F430 is reduced to the nickel(I) oxidation state by titanium(III) citrate.

The nickel porphinoid, coenzyme F430, is the prosthetic group of methyl-coenzyme M reductase. The active form of the enzyme exhibits Ni-EPR signals designated as MCR-red1 and MCR-red2. The inactive form of the enzyme is either EPR silent or it exhibits a distinct Ni-EPR signal designated MCR-ox1. Evidence is presented here that the MCR-ox1 form of the enzyme can be converted in vitro to the MCR-red1 form by reduction with titanium(III) citrate at pH 9. During conversion, the specific activity increases with increasing MCR-red1 spin concentration from 2 U/mg to approximately 100 U/mg at spin concentrations higher than 80%. The reduced methyl-coenzyme-M reductase shows an ultraviolet/visible spectrum characteristic for coenzyme F430 in the Ni(I) oxidation state, with maxima at 386 nm and at 750 nm. The results indicate that methyl-coenzyme-M reductase is activated when the enzyme-bound coenzyme F430 is reduced to the Ni(I) oxidation state. The experiments were performed with purified methyl-coenzyme-M reductase isoenzyme I of Methanobacterium thermoautotrophicum (strain Marburg).

Bacterial Proteins↗

PubMed1997

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase.

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO2 reduction to CH4 contain either an NADP-dependent or a coenzyme F420-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N5, N10-methylenetetrahydromethanopterin dehydrogenase and the N5, N10-methylenetetrahydromethanopterin reductase, two enzymes involved in CO2 reduction to CH4, are specific for F420. This raised the question how F420H2 is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F420 with NADPH. The F420-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced.

Alcohol Oxidoreductases↗

PubMed1997

A selenium-dependent and a selenium-independent formylmethanofuran dehydrogenase and their transcriptional regulation in the hyperthermophilic Methanopyrus kandleri.

The genome of Methanopyrus kandleri was found to harbour a gene, fwuB, predicted to encode the catalytic subunit of a tungsten formylmethanofuran dehydrogenase with an active site selenocysteine, and a second gene, fwcB, encoding a tungsten formylmethanofuran dehydrogenase with an active site cysteine. Northern blot and primer-extension analysis revealed that both genes were differentially transcribed. During growth of the methanogen on medium supplemented with selenium only fwuB was transcribed, whereas transcription of both fwuB and fwcB was observed on selenium-deprived medium. Growth of M. kandleri was stimulated by tungstate and selenite but not by molybdate. The findings indicate that the hyperthermophilic archaeon contains two tungsten isoenzymes of formylmethanofuran dehydrogenase, one of which is a novel selenium enzyme. They also indicate that the hyperthermophilic methanogen probably does not contain a molybdenum formylmethanofuran dehydrogenase which appears to be present only in thermophilic and mesophilic methanogens.

Aldehyde Oxidoreductases↗

PubMed1997

Crystallization and preliminary X-ray diffraction studies of methyl-coenzyme M reductase from methanobacterium thermoautotrophicum.

Methyl-coenzyme M reductase isoenzyme I from the methanogenic Archaeon, Methanobacterium thermoautotrophicum (strain Marburg), was crystallized by vapor diffusion methods. Crystal form M obtained with 2-methyl-2,4-pentanediol as the precipitant displayed space group P2(1), with unit cell parameters of a=83.2 A, b=117.4 A, c=125.1 A, and beta= 92.6 degrees, and diffracted at better than 2.8 A resolution. Crystal form P grown from polyethylene glycol 400 belonged to space group P2(1), and had unit cell parameters of a=83.1 A, b=120.2 A, c=123.1 A, and beta=91.7 degrees, diffracting at least to 1.7 A resolution. Both crystal forms have one molecule per asymmetric unit and are suitable for X-ray structure analysis.

Crystallization↗