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Rudolf K Thauer - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of the apoenzyme of the iron sulphur cluster free hydrogenase
Journal of Molecular Biology, 2006Co-Authors: Oliver Pilak, Seigo Shima, Bjorn Mamat, Sonja Vogt, Christoph H Hagemeier, Rudolf K Thauer, Clemens Vonrhein, Eberhard Warkentin, Ulrich ErmlerAbstract:The iron-sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor. The iron is coordinated by two CO molecules, one sulphur and a pyridone derivative, which is linked via a phosphodiester bond to a guanosine base. We report here on the crystal structure of the Hmd apoenzyme from Methanocaldococcus jannaschii at 1.75 A and from Methanopyrus kandleri at 2.4 A resolution. Homodimeric Hmd reveals a unique architecture composed of one central and two identical peripheral globular units. The central unit is composed of the intertwined C-terminal segments of both subunits, forming a novel intersubunit fold. The two peripheral units consist of the N-terminal domain of each subunit. The Rossmann fold-like structure of the N-terminal domain contains a mononucleotide-binding site, which could harbour the GMP moiety of the cofactor. Another binding site for the iron-containing cofactor is most probably Cys176, which is located at the bottom of a deep intersubunit cleft and which has been shown to be essential for enzyme activity. Adjacent to the iron of the cofactor modelled as a ligand to Cys176, an extended U-shaped extra electron density, interpreted as a polyethyleneglycol fragment, suggests a binding site for the substrate methenyltetrahydromethanopterin.
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coenzyme f420 dependent methylenetetrahydromethanopterin dehydrogenase mtd from Methanopyrus kandleri a methanogenic enzyme with an unusual quarternary structure
Journal of Molecular Biology, 2003Co-Authors: Christoph H Hagemeier, Seigo Shima, Rudolf K Thauer, Gleb Bourenkov, Hans D Bartunik, Ulrich ErmlerAbstract:The fourth reaction step of CO(2)-reduction to methane in methanogenic archaea is catalyzed by coenzyme F(420)-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd). We have structurally characterized this enzyme in the selenomethionine-labelled form from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri at 1.54A resolution using the single wavelength anomalous dispersion method for phase determination. Mtd was found to be a homohexameric protein complex that is organized as a trimer of dimers. The fold of the individual subunits is composed of two domains: a larger alpha,beta domain and a smaller helix bundle domain with a short C-terminal beta-sheet segment. In the homohexamer the alpha,beta domains are positioned at the outside of the enzyme, whereas, the helix bundle domains assemble towards the inside to form an unusual quarternary structure with a 12-helix bundle around a 3-fold axis. No structural similarities are detectable to other enzymes with F(420) and/or substituted tetrahydropterins as substrates. The substrate binding sites of F(420) and methylenetetrahydromethanopterin are most likely embedded into a crevice between the domains of one subunit, their isoalloxazine and tetrahydropterin rings being placed inside a pocket formed by this crevice and a loop segment of the adjacent monomer of the dimer. Mtd revealed the highest stability at low salt concentrations of all structurally characterized enzymes from M.kandleri. This finding might be due to the compact quaternary structure that buries 36% of the monomer surface and to the large number of ion pairs.
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coenzyme f420 dependent methylenetetrahydromethanopterin dehydrogenase from Methanopyrus kandleri the selenomethionine labelled and non labelled enzyme crystallized in two different forms
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Christoph H Hagemeier, Seigo Shima, Rudolf K Thauer, Eberhard Warkentin, Ulrich ErmlerAbstract:Coenzyme F(420)-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) is an enzyme involved in methanogenic energy metabolism which reversibly catalyzes the reduction of methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) to methylenetetrahydromethanopterin (methylene-H(4)MPT). The enzyme from the hyperthermophilic methanoarchaeon Methanopyrus kandleri could be crystallized: the non-labelled enzyme had unit-cell parameters a = 119.1, b = 151.0, c = 219.4 A and space group C222(1), while the selenomethionine-labelled enzyme had unit-cell parameters a = 119.6, b = 151.0, c = 109.9 A and also belonged to space group C222(1), indicating a surprising bisection of the c axis. The crystals grown from the non-labelled and labelled enzyme contained six and three monomers in the asymmetric unit and diffracted to about 1.9 and 1.5 A, respectively. The crystal packing of the two crystal forms seems to be similar. In particular, the crystals of the selenomethionine-labelled enzyme are highly suitable for X-ray structure determination.
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tetrahydromethanopterin specific enzymes from Methanopyrus kandleri
Methods in Enzymology, 2001Co-Authors: Seigo Shima, Rudolf K ThauerAbstract:Publisher Summary Methanopyrus kandleri is a hyperthermophilic archaeon growing optimally at 98° on H 2 and CO 2 with the formation of CH 4 . The organism belonging to the kingdom of Euryarchaeota is the most thermophilic methanogen known so far and is phylogenetically only distantly related to all other known methanogens. The pathway of CO 2 reduction to CH 4 in M. kandleri has been shown to be identical to that used in all other methanogens. It involves six tetrahydromethanopterin-specific enzymes. Tetrahydromethanopterin (H 4 MPT) is a tetrahydrofolate analog. This chapter describes the purification, assay, and properties of the five characterized H 4 MPT-specific enzymes from M. kandleri . It also provides a description of the isolation of the coenzymes required to assay these enzymes.
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identification of the active site histidine in the corrinoid protein mtra of the energy conserving methyltransferase complex from methanobacterium thermoautotrophicum
FEBS Journal, 1997Co-Authors: Ulrike Harms, Rudolf K ThauerAbstract: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 froin M. thermoautotrophicum, Methanococcus jannaschii, and Methanopyrus kandleri and of MtxA from Methanosarcina barkeri showing that only His84 is conserved. For comparison, the DNA sequences of the mtrEDCBAGH operon in M. kundleri and of the mtxXAH operon in M. barkeri were determined.
Dieter Söll - One of the best experts on this subject based on the ideXlab platform.
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archaeal 3 phosphate rna splicing ligase characterization identifies the missing component in trna maturation
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Markus Englert, Kelly Sheppard, Aaron Aslanian, John R Yates, Dieter SöllAbstract:Intron removal from tRNA precursors involves cleavage by a tRNA splicing endonuclease to yield tRNA 3′-halves beginning with a 5′-hydroxyl, and 5′-halves ending in a 2′,3′-cyclic phosphate. A tRNA ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. Although this 3′-P RNA splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. Here we report the purification of this ligase from Methanopyrus kandleri cells, and its assignment to the still uncharacterized RtcB protein family. Studies with recombinant Pyrobaculum aerophilum RtcB showed that the enzyme is able to join spliced tRNA halves to mature-sized tRNAs where the joining phosphodiester linkage contains the phosphate originally present in the 2′,3′-cyclic phosphate. The data confirm RtcB as the archaeal RNA 3′-P ligase. Structural genomics efforts previously yielded a crystal structure of the Pyrococcus horikoshii RtcB protein containing a new protein fold and a conserved putative Zn2+ binding cleft. This structure guided our mutational analysis of the P. aerophilum enzyme. Mutations of highly conserved residues in the cleft (C100A, H205A, H236A) rendered the enzyme inactive suggesting these residues to be part of the active site of the P. aerophilum ligase. There is no significant sequence similarity between the active sites of P. aerophilum ligase and that of T4 RNA ligase, nor ligases from plants and fungi. RtcB sequence conservation in archaea and in eukaryotes implicates eukaryotic RtcB as the long-sought animal 3′-P RNA ligase.
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the heteromeric nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge helix bulge motifs of joined trna halves
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Lennart Randau, Mircea Podar, Katherine Calvin, Michelle Hall, Jing Yuan, Dieter SöllAbstract:Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5' and 3' tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge-helix-bulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate.
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Cysteinyl-tRNACys Formation in Methanocaldococcus jannaschii: the Mechanism Is Still Unknown
Journal of bacteriology, 2004Co-Authors: Benfang Ruan, Hiroaki Nakano, Masashi Tanaka, Jonathan A. Mills, Joseph A. Devito, Bokkee Min, K. Brooks Low, John R. Battista, Dieter SöllAbstract:Most organisms form Cys-tRNACys, an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. Soll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crecy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the “missing” CysRS activity for in vivo Cys-tRNACys formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the Km for cysteine and a ninefold reduction in the kcat for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNACys synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNACys formation in M. jannaschii still remains to be discovered.
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the structural basis of cysteine aminoacylation of trnapro by prolyl trna synthetases
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Satwik Kamtekar, Dieter Söll, W D Kennedy, Jimin Wang, Constantinos Stathopoulos, Thomas A SteitzAbstract:Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, or Methanopyrus kandleri. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 A: apo M. jannaschii ProRS, and M. thermautotrophicus ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl- and prolyl-adenylate analogues have similar, nanomolar affinities for M. thermautotrophicus ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3'-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that M. jannaschii ProRS misacylates tRNA(Pro) with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both tRNA(Pro) and tRNA(Cys) with their cognate amino acids.
Seigo Shima - One of the best experts on this subject based on the ideXlab platform.
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Post-translational modifications in the active site region of methyl-coenzyme M reductase from methanogenic and methanotrophic archaea
FEBS Journal, 2007Co-Authors: Jörg Kahnt, Bärbel Buchenau, Felix Mahlert, Martin Krüger, Seigo ShimaAbstract:Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. Isoenzyme I from Methanothermobacter marburgensiswas shown to contain a thioxo peptide bond and four methylated amino acids in the active site region. We report here that MCRs from all methanogens investigated contain the thioxo peptide bond, but that the enzymes differ in their post-translational methylations. The MS analysis included MCR I and MCR II from Methanothermobacter marburgensis, MCR I from Methanocaldococcus jannaschii and Methanoculleus thermophilus, and MCR from Methanococcus voltae, Methanopyrus kandleri and Methanosarcina barkeri. Two MCRs isolated from Black Sea mats containing mainly methanotrophic archaea of the ANME-1 cluster were also analyzed.
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the crystal structure of the apoenzyme of the iron sulphur cluster free hydrogenase
Journal of Molecular Biology, 2006Co-Authors: Oliver Pilak, Seigo Shima, Bjorn Mamat, Sonja Vogt, Christoph H Hagemeier, Rudolf K Thauer, Clemens Vonrhein, Eberhard Warkentin, Ulrich ErmlerAbstract:The iron-sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor. The iron is coordinated by two CO molecules, one sulphur and a pyridone derivative, which is linked via a phosphodiester bond to a guanosine base. We report here on the crystal structure of the Hmd apoenzyme from Methanocaldococcus jannaschii at 1.75 A and from Methanopyrus kandleri at 2.4 A resolution. Homodimeric Hmd reveals a unique architecture composed of one central and two identical peripheral globular units. The central unit is composed of the intertwined C-terminal segments of both subunits, forming a novel intersubunit fold. The two peripheral units consist of the N-terminal domain of each subunit. The Rossmann fold-like structure of the N-terminal domain contains a mononucleotide-binding site, which could harbour the GMP moiety of the cofactor. Another binding site for the iron-containing cofactor is most probably Cys176, which is located at the bottom of a deep intersubunit cleft and which has been shown to be essential for enzyme activity. Adjacent to the iron of the cofactor modelled as a ligand to Cys176, an extended U-shaped extra electron density, interpreted as a polyethyleneglycol fragment, suggests a binding site for the substrate methenyltetrahydromethanopterin.
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coenzyme f420 dependent methylenetetrahydromethanopterin dehydrogenase mtd from Methanopyrus kandleri a methanogenic enzyme with an unusual quarternary structure
Journal of Molecular Biology, 2003Co-Authors: Christoph H Hagemeier, Seigo Shima, Rudolf K Thauer, Gleb Bourenkov, Hans D Bartunik, Ulrich ErmlerAbstract:The fourth reaction step of CO(2)-reduction to methane in methanogenic archaea is catalyzed by coenzyme F(420)-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd). We have structurally characterized this enzyme in the selenomethionine-labelled form from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri at 1.54A resolution using the single wavelength anomalous dispersion method for phase determination. Mtd was found to be a homohexameric protein complex that is organized as a trimer of dimers. The fold of the individual subunits is composed of two domains: a larger alpha,beta domain and a smaller helix bundle domain with a short C-terminal beta-sheet segment. In the homohexamer the alpha,beta domains are positioned at the outside of the enzyme, whereas, the helix bundle domains assemble towards the inside to form an unusual quarternary structure with a 12-helix bundle around a 3-fold axis. No structural similarities are detectable to other enzymes with F(420) and/or substituted tetrahydropterins as substrates. The substrate binding sites of F(420) and methylenetetrahydromethanopterin are most likely embedded into a crevice between the domains of one subunit, their isoalloxazine and tetrahydropterin rings being placed inside a pocket formed by this crevice and a loop segment of the adjacent monomer of the dimer. Mtd revealed the highest stability at low salt concentrations of all structurally characterized enzymes from M.kandleri. This finding might be due to the compact quaternary structure that buries 36% of the monomer surface and to the large number of ion pairs.
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coenzyme f420 dependent methylenetetrahydromethanopterin dehydrogenase from Methanopyrus kandleri the selenomethionine labelled and non labelled enzyme crystallized in two different forms
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Christoph H Hagemeier, Seigo Shima, Rudolf K Thauer, Eberhard Warkentin, Ulrich ErmlerAbstract:Coenzyme F(420)-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) is an enzyme involved in methanogenic energy metabolism which reversibly catalyzes the reduction of methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) to methylenetetrahydromethanopterin (methylene-H(4)MPT). The enzyme from the hyperthermophilic methanoarchaeon Methanopyrus kandleri could be crystallized: the non-labelled enzyme had unit-cell parameters a = 119.1, b = 151.0, c = 219.4 A and space group C222(1), while the selenomethionine-labelled enzyme had unit-cell parameters a = 119.6, b = 151.0, c = 109.9 A and also belonged to space group C222(1), indicating a surprising bisection of the c axis. The crystals grown from the non-labelled and labelled enzyme contained six and three monomers in the asymmetric unit and diffracted to about 1.9 and 1.5 A, respectively. The crystal packing of the two crystal forms seems to be similar. In particular, the crystals of the selenomethionine-labelled enzyme are highly suitable for X-ray structure determination.
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tetrahydromethanopterin specific enzymes from Methanopyrus kandleri
Methods in Enzymology, 2001Co-Authors: Seigo Shima, Rudolf K ThauerAbstract:Publisher Summary Methanopyrus kandleri is a hyperthermophilic archaeon growing optimally at 98° on H 2 and CO 2 with the formation of CH 4 . The organism belonging to the kingdom of Euryarchaeota is the most thermophilic methanogen known so far and is phylogenetically only distantly related to all other known methanogens. The pathway of CO 2 reduction to CH 4 in M. kandleri has been shown to be identical to that used in all other methanogens. It involves six tetrahydromethanopterin-specific enzymes. Tetrahydromethanopterin (H 4 MPT) is a tetrahydrofolate analog. This chapter describes the purification, assay, and properties of the five characterized H 4 MPT-specific enzymes from M. kandleri . It also provides a description of the isolation of the coenzymes required to assay these enzymes.
Ulrich Ermler - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of the apoenzyme of the iron sulphur cluster free hydrogenase
Journal of Molecular Biology, 2006Co-Authors: Oliver Pilak, Seigo Shima, Bjorn Mamat, Sonja Vogt, Christoph H Hagemeier, Rudolf K Thauer, Clemens Vonrhein, Eberhard Warkentin, Ulrich ErmlerAbstract:The iron-sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor. The iron is coordinated by two CO molecules, one sulphur and a pyridone derivative, which is linked via a phosphodiester bond to a guanosine base. We report here on the crystal structure of the Hmd apoenzyme from Methanocaldococcus jannaschii at 1.75 A and from Methanopyrus kandleri at 2.4 A resolution. Homodimeric Hmd reveals a unique architecture composed of one central and two identical peripheral globular units. The central unit is composed of the intertwined C-terminal segments of both subunits, forming a novel intersubunit fold. The two peripheral units consist of the N-terminal domain of each subunit. The Rossmann fold-like structure of the N-terminal domain contains a mononucleotide-binding site, which could harbour the GMP moiety of the cofactor. Another binding site for the iron-containing cofactor is most probably Cys176, which is located at the bottom of a deep intersubunit cleft and which has been shown to be essential for enzyme activity. Adjacent to the iron of the cofactor modelled as a ligand to Cys176, an extended U-shaped extra electron density, interpreted as a polyethyleneglycol fragment, suggests a binding site for the substrate methenyltetrahydromethanopterin.
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coenzyme f420 dependent methylenetetrahydromethanopterin dehydrogenase mtd from Methanopyrus kandleri a methanogenic enzyme with an unusual quarternary structure
Journal of Molecular Biology, 2003Co-Authors: Christoph H Hagemeier, Seigo Shima, Rudolf K Thauer, Gleb Bourenkov, Hans D Bartunik, Ulrich ErmlerAbstract:The fourth reaction step of CO(2)-reduction to methane in methanogenic archaea is catalyzed by coenzyme F(420)-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd). We have structurally characterized this enzyme in the selenomethionine-labelled form from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri at 1.54A resolution using the single wavelength anomalous dispersion method for phase determination. Mtd was found to be a homohexameric protein complex that is organized as a trimer of dimers. The fold of the individual subunits is composed of two domains: a larger alpha,beta domain and a smaller helix bundle domain with a short C-terminal beta-sheet segment. In the homohexamer the alpha,beta domains are positioned at the outside of the enzyme, whereas, the helix bundle domains assemble towards the inside to form an unusual quarternary structure with a 12-helix bundle around a 3-fold axis. No structural similarities are detectable to other enzymes with F(420) and/or substituted tetrahydropterins as substrates. The substrate binding sites of F(420) and methylenetetrahydromethanopterin are most likely embedded into a crevice between the domains of one subunit, their isoalloxazine and tetrahydropterin rings being placed inside a pocket formed by this crevice and a loop segment of the adjacent monomer of the dimer. Mtd revealed the highest stability at low salt concentrations of all structurally characterized enzymes from M.kandleri. This finding might be due to the compact quaternary structure that buries 36% of the monomer surface and to the large number of ion pairs.
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coenzyme f420 dependent methylenetetrahydromethanopterin dehydrogenase from Methanopyrus kandleri the selenomethionine labelled and non labelled enzyme crystallized in two different forms
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Christoph H Hagemeier, Seigo Shima, Rudolf K Thauer, Eberhard Warkentin, Ulrich ErmlerAbstract:Coenzyme F(420)-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) is an enzyme involved in methanogenic energy metabolism which reversibly catalyzes the reduction of methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) to methylenetetrahydromethanopterin (methylene-H(4)MPT). The enzyme from the hyperthermophilic methanoarchaeon Methanopyrus kandleri could be crystallized: the non-labelled enzyme had unit-cell parameters a = 119.1, b = 151.0, c = 219.4 A and space group C222(1), while the selenomethionine-labelled enzyme had unit-cell parameters a = 119.6, b = 151.0, c = 109.9 A and also belonged to space group C222(1), indicating a surprising bisection of the c axis. The crystals grown from the non-labelled and labelled enzyme contained six and three monomers in the asymmetric unit and diffracted to about 1.9 and 1.5 A, respectively. The crystal packing of the two crystal forms seems to be similar. In particular, the crystals of the selenomethionine-labelled enzyme are highly suitable for X-ray structure determination.
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formylmethanofuran tetrahydromethanopterin formyltransferase from Methanopyrus kandleri new insights into salt dependence and thermostability
Structure, 1997Co-Authors: Ulrich Ermler, Rudolf K Thauer, Michael C Merckel, Seigo ShimaAbstract:Abstract Background: Formylmethanofuran: tetrahydromethanopterin formyltransferase (Ftr) from the methanogenic Archaeon Methanopyrus kandleri (optimum growth temperature 98°C) is a hyperthermophilic enzyme that is absolutely dependent on the presence of lyotropic salts for activity and thermostability. The enzyme is involved in the pathway of carbon dioxide reduction to methane and catalyzes the transfer of formyl from formylmethanofuran to tetrahydromethanopterin. Results: The crystal structure of Ftr, determined to a resolution of 1:73 A, reveals a homotetramer composed essentially of two dimers. Each subunit is subdivided into two tightly associated lobes both consisting of a predominantly antiparallel β sheet flanked by α helices forming an α / β sandwich structure. The approximate location of the active site was detected in a region close to the dimer interface. Conclusions: The adaptation of Ftr against high lyotropic salt concentrations is structurally reflected by a large number of negatively charged residues and their high local concentration on the surface of the protein. The salt-dependent thermostability of Ftr might be explained on a molecular basis by ionic interactions at the protein surface, involving both protein and inorganic salt ions, and the mainly hydrophobic interactions between the subunits and within the core.
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crystallization and preliminary x ray diffraction studies of formylmethanofuran tetrahydromethanopterin formyltransferase from Methanopyrus kandleri
Proteins, 1996Co-Authors: Seigo Shima, Rudolf K Thauer, Hartmut Michel, Ulrich ErmlerAbstract:Formylmethanofuran:tetrahydromethanopterin formyltransferase from the hyperthermophilic methanogenic Archaeon Methanopyrus kandleri (growth temperature optimum 98 degrees C) was crystallized by vapor diffusion methods. Crystal form M obtained with 2-methyl-2,4-pentanediol as precipitant displayed the space group P2(1) with unit cell parameters of a = 87.0 A, b = 75.4 A, c = 104.7 A, and beta = 113.9 degrees and diffracted better than 2 A resolution. Crystal form P grown from polyethylene glycol 8000 belonged to the space group I4(1)22 and had unit cell parameters of 157.5 A and 242.1 A. Diffraction data to 1.73 A were recorded. Crystal form S which was crystallized from (NH4)2SO4 in the space group I4(1)22 with unit cell parameters of 151.3 A and 249.5 A diffracted at least to 2.2 A resolution. All crystal forms probably have four molecules per asymmetric unit and are suitable for X-ray structure analysis.
Karl O Stetter - One of the best experts on this subject based on the ideXlab platform.
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the recombinant thermosome from the hyperthermophilic archaeon Methanopyrus kandleri in vitro analysis of its chaperone activity
Biological Chemistry, 1999Co-Authors: T. Minuth, Karl O Stetter, Gerhard Frey, Stefan Andra, Martina Henn, Kerstin Rutkat, Reinhard Rachel, Rainer JaenickeAbstract:The archaeon Methanopyrus kandleri is the most thermophilic methanogen presently known. It contains a chaperonin (thermosome) which represents a 951 kDa homo-hexadecameric protein complex with NH4+-dependent ATPase activity. Since its synthesis is not increased upon heat shock, we set out to test its chaperone function. In order to obtain the chaperonin in amounts sufficient for functional investigations, the gene encoding the 60 kDa subunit was expressed in E. coili BL21 (DE3) cells. Purification yielded soluble, high-molecular-mass double-ring complexes, indistinguishable from the natural thermosome. In order to study the functional properties of the recombinant protein complex, pig citrate synthase, yeast alcohol dehydrogenase, yeast alpha-glucosidase, bovine insulin, and Thermotoga phosphoglycerate kinase were used as model substrates. The results demonstrate that the recombinant M. kandleri thermosome possesses a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins after chemical denaturation. However, the chaperonin only forms dead-end complexes with its non-native substrates, no release is detectable at temperatures between 25 and 60 degrees C.
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the thermosome from Methanopyrus kandleri possesses an nh es rb ei4 rb dependent atpase activity
FEBS Journal, 1998Co-Authors: Stefan Andra, Rainer Jaenicke, Gerhard Frey, Karl O StetterAbstract:The ATPase activity of the thermosome from a methanogen, Methanopyrus kandleri, was characterized in detail. In contrast to all other known chaperonins, enzymatic ATP hydrolysis was found to be strictly dependent on high levels of ammonium salts in vitro. The ths gene encoding the thermosome subunit from the hyperthermophilic M. kandleri was functionally expressed in Escherichia coli and the overproduced polypeptide was assembled into intact thermosome complexes in the mesophilic host. The recombinant particles could be purified by a simple two-step procedure including only one chromatographic step. Structural and biochemical properties of the recombinant protein were closely similar to those of the natural complex. Western blot analysis with an antiserum against the M. kandleri thermosome indicated the presence of at least two subfamilies of archaeal chaperonins.
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purification and structural characterization of the thermosome from the hyperthermophilic archaeum Methanopyrus kandleri
FEBS Letters, 1996Co-Authors: Stefan Andra, Wolfgang Baumeister, Gerhard Frey, Michael Nitsch, Karl O StetterAbstract:From Methanopyrus kandleri, the most thermophilic methanogen known so far, we have purified to homogeneity a protein complex of high molecular mass. Image analysis of transmission electron micrographs revealed a barrel-shaped particle composed of two rings with 8-fold symmetry. Only one type of subunit could be detected. The corresponding gene has been cloned and sequenced. The deduced amino acid sequence shows high homology with the members of group II chaperonins. The structure of the projection and the sequence homology suggest that this particle is the first thermosome isolated from a methanogen.
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a novel unsaturated archaeal ether core lipid from the hyperthermophile Methanopyrus kandleri
Systematic and Applied Microbiology, 1993Co-Authors: Doris Hafenbradl, Martin Keller, Ralf Thiericke, Karl O StetterAbstract:Summary From cells of the hyperthermophilic Methanopyrus kandleri , a novel unsaturated core ether lipid was isolated. By UV-, IR-, Mass- and 13 C NMR-spectroscopy it was identified as 2,3-di-O-geranylgeranyl- sn -glycerol. In addition, the cells contain geranylgeraniol. Under the view of evolution, the presence of a terpenoid lipid is a rather primitive feature. This is in line with the deep short lineage of Methanopyrus within the 16S rRNA-based phylogenetic tree.
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salt dependence kinetic properties and catalytic mechanism of n formylmethanofuran tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri
FEBS Journal, 1992Co-Authors: Jurgen Breitung, Karl O Stetter, Dietmar Linder, Gerhard Borner, Sabine Scholz, Rudolf K ThauerAbstract:N-Formylmethanofuran(CHO-MFR): tetrahydromethanopterin(H4MPT) formyltransferase (for-myltransferase) 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 revcaled 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°C, the Vmax was 2700 U/mg (1 U = 1 μmol/min), the Km for CHO-MFR was 50 μM and the Km for H4MPT was 100 μM. At 90°C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65°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°C decreased in the order K2HPO4= (NH4)2SO4≫ KCI = 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.