Showing papers on "Methanosarcina barkeri published in 2002"
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TL;DR: Results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid to be encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri.
Abstract: Pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is thepylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to known lysyl-tRNA synthetases. Homologs of pylS and pylT are found in a Gram-positive bacterium. Charging a tRNACUA with lysine is a likely first step in translating UAG amber codons as pyrrolysine in certain methanogens. Our results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid.
568 citations
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TL;DR: The UAG-encoded residue in a 1.55 angstrom resolution structure of the Methanosarcina barkerimonomethylamine methyltransferase (MtmB) reveals a homohexamer comprised of individual subunits with a TIM barrel fold that appears consistent with a lysine in amide-linkage to (4R,5R)-4-substituted-pyrroline-5-carboxylate.
Abstract: Genes encoding methanogenic methylamine methyltransferases all contain an in-frame amber (UAG) codon that is read through during translation. We have identified the UAG-encoded residue in a 1.55 angstrom resolution structure of the Methanosarcina barkeri monomethylamine methyltransferase (MtmB). This structure reveals a homohexamer comprised of individual subunits with a TIM barrel fold. The electron density for the UAG-encoded residue is distinct from any of the 21 natural amino acids. Instead it appears consistent with a lysine in amide-linkage to (4R,5R)-4-substituted-pyrroline-5-carboxylate. We suggest that this amino acid be named l-pyrrolysine.
373 citations
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TL;DR: The ability of methanogens to interact with extracellular quinones, humic acids and Fe(III) oxides raises the possibility that this functional group of organ-isms contributes to Fe( III) and humic acid reduction under certain conditions in the environment and provides an alternative explanation for the inhibition of meethanogenesis in some Fe(II)-containing ecosystems.
Abstract: Summary Five methanogens (Methanosarcina barkeri MS, Methanosphaera cuniculi 1R7, Methanobacterium palustre F, Methanococcus voltaei A3 and Methanolobus vulcani PL-12/M) were investigated for their ability to reduce Fe(III) oxide and the soluble quinone anthraquinone-2,6-disulphonate (AQDS). Two species (M. barkeri and M. voltaei) reduced significant amounts of Fe(III) oxide using hydrogen as the electron donor, and 0.1 mM AQDS greatly accelerated Fe(III) reduction by these organisms. Although Fe(III) appeared to inhibit growth and methanogenesis of some strains, hydrogen partial pressures under donor-limited conditions were much lower (<0.5 Pa) in the presence of Fe(III) than in normal media (1‐10 Pa) for all species except for M. vulcani. These results demonstrate that electrons were transferred to Fe(III) by hydrogen-utilizing methanogens even when growth and methanogenesis were inhibited. All species except the obligate methylotroph M. vulcani were able to reduce AQDS when their growth substrates were present as electron donors, and rates were highest when organisms used hydrogen as the electron donor. Purified soil humic acids could also be reduced by the AQDS-reducing methanogens. The ability of methanogens to interact with extracellular quinones, humic acids and Fe(III) oxides raises the possibility that this functional group of organisms contributes to Fe(III) and humic acid reduction under certain conditions in the environment and provides an alternative explanation for the inhibition of methanogenesis in some Fe(III)-containing ecosystems.
218 citations
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TL;DR: Results obtained from growth experiments, cell suspension experiments, and enzyme activity measurements in cell extracts provide compelling evidence for essential functions of Ech and a 2[4Fe-4S] ferredoxin in the metabolism of M. barkeri.
Abstract: Ech hydrogenase (Ech) from the methanogenic archaeon Methanosarcina barkeri catalyzes the reversible reduction of ferredoxin by H2 and is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). To elucidate the physiological role(s) of Ech a mutant lacking this enzyme was constructed. The mutant was unable to grow on methanol/H2/CO2, H2/CO2, or acetate as carbon and energy sources but showed wild-type growth rates with methanol as sole substrate. Addition of pyruvate to the growth medium restored growth on methanol/H2/CO2 but not on H2/CO2 or acetate. Results obtained from growth experiments, cell suspension experiments, and enzyme activity measurements in cell extracts provide compelling evidence for essential functions of Ech and a 2[4Fe-4S] ferredoxin in the metabolism of M. barkeri. The following conclusions were made. (i) In acetoclastic methanogenesis, Ech catalyzes H2 formation from reduced ferredoxin, generated by the oxidation of the carbonyl group of acetate to CO2. (ii) Under autotrophic growth conditions, the enzyme catalyzes the energetically unfavorable reduction of ferredoxin by H2, most probably driven by reversed electron transport, and the reduced ferredoxin thus generated functions as low potential electron donor for the synthesis of pyruvate in an anabolic pathway. (iii) Reduced ferredoxin in addition provides the reducing equivalents for the first step of methanogenesis from H2/CO2, the reduction of CO2 to formylmethanofuran. Thus, in vivo genetic analysis has led to the identification of the electron donor of this key initial step of methanogenesis.
175 citations
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TL;DR: The purification of a multisubunit membrane-bound enzyme complex from A. fulgidus that contains a subunit related to the catalytic subunit of Hdr that may have a similar active site and a similar catalytic function is reported on.
Abstract: Heterodisulfide reductase (Hdr) is a unique disulfide reductase that plays a key role in the energy metabolism of methanogenic archaea. The genome of the sulfate-reducing archaeon Archaeoglobus fulgidus encodes several proteins of unknown function with high sequence similarity to the catalytic subunit of Hdr. Here we report on the purification of a multisubunit membrane-bound enzyme complex from A. fulgidus that contains a subunit related to the catalytic subunit of Hdr. The purified enzyme is a heme/iron-sulfur protein, as deduced by UV/Vis spectroscopy, EPR spectroscopy, and the primary structure. It is composed of four different subunits encoded by a putative transcription unit (AF499, AF501–AF503). A fifth protein (AF500) encoded by this transcription unit could not be detected in the purified enzyme preparation. Subunit AF502 is closely related to the catalytic subunit HdrD of Hdr from Methanosarcina barkeri. AF501 encodes a membrane-integral cytochrome, and AF500 encodes a second integral membrane protein. AF499 encodes an extracytoplasmic iron-sulfur protein, and AF503 encodes an extracytoplasmic c-type cytochrome with three heme c-binding motifs. All of the subunits show high sequence similarity to proteins encoded by the dsr locus of Allochromatium vinosum and to subunits of the Hmc complex from Desulfovibrio vulgaris. The heme groups of the enzyme are rapidly reduced by reduced 2,3-dimethyl-1,4-naphthoquinone (DMNH2), which indicates that the enzyme functions as a menaquinol–acceptor oxidoreductase. The physiological electron acceptor has not yet been identified. Redox titrations monitored by EPR spectroscopy were carried out to characterize the iron-sulfur clusters of the enzyme. In addition to EPR signals due to [4Fe-4S]+ clusters, signals of an unusual paramagnetic species with g values of 2.031, 1.994, and 1.951 were obtained. The paramagnetic species could be reduced in a one-electron transfer reaction, but could not be further oxidized, and shows EPR properties similar to those of a paramagnetic species recently identified in Hdr. In Hdr this paramagnetic species is specifically induced by the substrates of the enzyme and is thought to be an intermediate of the catalytic cycle. Hence, Hdr and the A. fulgidus enzyme not only share sequence similarity, but may also have a similar active site and a similar catalytic function.
63 citations
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TL;DR: Initial studies suggest that pyrrolysine may be co-translationally inserted during protein synthesis, probably by a mechanism analogous to that operating during selenocysteine incorporation.
61 citations
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TL;DR: Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential and the possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed.
Abstract: The purified membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri was studied with electron paramagnetic resonance (EPR) focusing on the properties of the iron-sulphur clusters. The EPR spectra showed signals from three different [4Fe-4S] clusters. Two of the clusters could be reduced under 101 kPa of H2, whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni-Fe site indicated that this was the proximal cluster as found in all [NiFe]-hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe-4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential. The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed.
35 citations
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TL;DR: Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes of M. mazei Gö1 showed that they are most closely related to Methanosarcina barkeri nif2 genes, and also closely resemble those for the corresponding nif products of the gram-positive bacterium C. acetobutylicum.
Abstract: The mesophilic methanogenic archaeon Methanosarcina mazei strain Go1 is able to utilize molecular nitrogen (N2) as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif) gene cluster in M. mazei Go1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities to nifH, nifI1, nifI2, nifD, nifK, nifE and nifN, similar to other diazotrophic methanogens and certain bacteria such as Clostridium acetobutylicum, with the two glnB-like genes (nifI1 and nifI2) located between nifH and nifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes of M. mazei Go1 showed that they are most closely related to Methanosarcina barkeri nif2 genes, and also closely resemble those for the corresponding nif products of the gram-positive bacterium C. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that the M. mazei nif genes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of the M. mazei nifH gene, which may have a function in transcriptional regulation of the nif operon.
32 citations
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TL;DR: Zinc XAFS (X-ray absorption fine structure) results indicating that, in the absence of coenzyme M, zinc is probably coordinated by a single sulfur ligand and three oxygen or nitrogen ligands.
Abstract: Methanol:coenzyme M methyltransferase from methanogenic archaea is a cobalamin-dependent enzyme composed of three different subunits: MtaA, MtaB and MtaC. MtaA is a zinc protein that catalyzes the methylation of coenzyme M (HS-CoM) with methylcob(III)alamin. We report zinc XAFS (X-ray absorption fine structure) results indicating that, in the absence of coenzyme M, zinc is probably coordinated by a single sulfur ligand and three oxygen or nitrogen ligands. In the presence of coenzyme M, one (N/O)-ligand was replaced by sulfur, most likely due to ligation of the thiol group of coenzyme M. Mutations in His237 or Cys239, which are proposed to be involved in ligating zinc, resulted in an over 90% loss in enzyme activity and in distinct changes in the zinc ligands. In the His237 → Ala and Cys239 → Ala mutants, coenzyme M also seemed to bind efficiently by ligation to zinc indicating that some aspects of the zinc ligand environment are surprisingly uncritical for coenzyme M binding.
29 citations
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TL;DR: The metabolic activities of both Desulfovibrio strain B and M. barkeri 227 were essential for the complete degradation of furfural.
Abstract: Methanogenesis from furfural by defined mixed cultures was studied. Under sulfate-reducing conditions, a Desulfovibrio strain was used as the furfural-degrading species producing acetic acid. This sulfate-reducing bacterium (SRB) Desulfovibrio strain B is an incomplete oxidizer, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. Introduction of acetate-utilizing methanogenic archaeon Methanosarcina barkeri 227 converted acetic acid to methane. This well-defined mixed consortium used furfural as its sole source of carbon and converted it to methane and CO(2). In the mixed culture, when a methanogen inhibitor was used in the culture medium, furfural was converted to acetic acid by the Desulfovibrio strain B, but acetic acid did not undergo further metabolism. On the other hand, when the growth of Desulfovibrio in the consortium was suppressed with a specific SRB inhibitor, namely molybdate, furfural was not degraded. Thus, the metabolic activities of both Desulfovibrio strain B and M. barkeri 227 were essential for the complete degradation of furfural.
12 citations
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TL;DR: Researchers at Ohio State University have discovered a 22nd genetically encoded amino acids in mammalian cells, and identify pyrrolysine in the 1.55-A crystal structure of monomethylamine methyltransferase from the microbe Methanosarcina barkeri.
Abstract: NATURE USES ONLY A HANDful of amino acid building blocks to generate immense complexity from a relatively simple genetic code. There are just 20 genetically encoded amino acids in mammalian cells; a few organisms use 21. Now, researchers at Ohio State University have discovered a 22nd [ Science , 296,1459 and 1462 (2002)]. The team, led by microbiology professor Joseph A. Krzycki and chemistry and biochemistry professor Michael K. Chan, calls the amino acid L-pyrrolysine. The novel residue is an amide-linked 4-substituted pyrroline-5-carboxylate lysine derivative. Chan and graduate student Bing Hao identified pyrrolysine in the 1.55-A crystal structure of monomethylamine methyltransferase (MtmB) from the microbe Methanosarcina barkeri , found at the bottom of freshwater lakes. An enzyme in the pathway that makes methane from methylamine, MtmB has a barrel protein fold with pyrrolysine tucked into the bottom. But finding a novel amino acid in a protein structure doesn't necessarily mean the residue is ...