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Methanosarcina barkeri

About: Methanosarcina barkeri is a research topic. Over the lifetime, 703 publications have been published within this topic receiving 32151 citations.


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Journal ArticleDOI
TL;DR: The enzyme from M. barkeri was found to contain, per mol, 1 mol iron, but no manganese – in agreement with the general observation that anaerobically growing organisms only contain iron superoxide dismutase.
Abstract: Methanosarcina barkeri is a methanogenic archaeon that can only grow under strictly anoxic conditions but which can survive oxidative stress. We have recently reported that the organism contains a monofunctional catalase. We describe here that it also possesses an active iron superoxide dismutase. The enzyme was purified in three steps over 130-fold in a 14% yield to a specific activity of 1500 U/mg. SDS-PAGE revealed the presence of only one band, at an apparent molecular mass of 25 kDa. The primary structure determined from the cloned and sequenced gene revealed similarity to iron- and manganese superoxide dismutases. The highest similarity was to the iron superoxide dismutase from Methanobacterium thermoautotrophicum. The enzyme from M. barkeri was found to contain, per mol, 1 mol iron, but no manganese – in agreement with the general observation that anaerobically growing organisms only contain iron superoxide dismutase. The enzyme was not inhibited by cyanide (10 mM), which is a property shared by all iron- and manganese superoxide dismutases. The presence of superoxide dismutase in M. barkeri is noteworthy since a gene encoding superoxide dismutase (sod) has not been found in Archaeoglobus fulgidus, a sulfate-reducing archaeon most closely related to the Methanosarcinaceae.

52 citations

Journal ArticleDOI
TL;DR: A mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed and proposed and is proposed for methanogenic archaebacteria.
Abstract: An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N5-methyltetrahydromethanopterin (CH3−H4MPT) to coenzyme M (H−S−CoM) in methanogenic archaebacteria. With this method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri and in H2/CO2-grown Methanobacterium thermoautotrophicum were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH3−S−CoM formation from CH3−H4MPT (apparent Km=50 μM) and H−S−CoM (apparent Km=250 μM) of approximately 0.6 μmol·min-1·mg protein-1. For activity the presence of Ti(III) citrate (apparent Km=15 μM) and of ATP (apparent Km=30 μM) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH3−H4MPT in the absence of H−S−CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH3−H4MPT to H−S−CoM in the absence of ATP and Ti(III). Demethylation in the presence of H−S−CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.

52 citations

Journal ArticleDOI
TL;DR: N5N10-Methylenetetrahydro-methanopterin reductase, an enzyme involved in methanogenesis from CO2, was purified from M. kandleri, a novel group of abyssal methanogenic archaebacteria that shows no close phylogenetic relationship to any methanogens known so far.
Abstract: Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H2 and CO2 and that shows no close phylogenetic relationship to any methanogens known so far. N 5 N 10 -Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogenesis from CO2, was purified from this hyperthermophile. The apparent molecular mass of the native enzyme was found to be 300 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only one polypeptide of apparent molecular mass 38 kDa. The ultraviolet/visible spectrum of the enzyme was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was specific for reduced coenzyme F420 as electron donor; NADH, NADPH or reduced dyes could not substitute for the 5-deazaflavin. The catalytic mechanism was found to be of the ternary complex type as deduced from initial velocity plots. V max at 65°C and pH 6.8 was 435 U/mg (kcat=275 s-1) and the K m for methylenetetrahydro-methanopterin and for reduced F420 were 6 μM and 4 μM, respectively. From Arrhenius plots an activation energy of 34 kJ/mol was determined. The Q 10 between 40°C and 90°C was 1.5. The reductase activity was found to be stimulated over 100-fold by sulfate and by phosphate. Maximal stimulation (100-fold) was observed at a sulfate concentration of 2.2 M and at a phosphate concentration of 2.5 M. Sodium-, potassium-, and ammonium salts of these anions were equally effective. Chloride, however, could not substitute for sulfate or phosphate in stimulating the enzyme activity. The thermostability of the reductase was found to be very low in the absence of salts. In their presence, however, the reductase was highly thermostable. Salt concentrations between 0.1 M and 1.5 M were required for maximal stability. Potassium salts proved more effective than ammonium salts, and the latter more effective than sodium salts in stabilizing the enzyme activity. The anion was of less importance. The N-terminal amino acid sequence of the reductase from M. kandleri was determined and compared with that of the enzyme from Methanobacterium thermoautotrophicum and Methanosarcina barkeri. Significant similarity was found.

52 citations

Journal ArticleDOI
TL;DR: A structural analysis of the lipid moiety of Methanosarcina spp. as discussed by the authors revealed that the hydroxydiether lipids were hydroxylated at position 3 of sn-2 phytanyl chains.
Abstract: Hydroxylated diether lipids are the most abundant lipids in Methanosarcina acetivorans, Methanosarcina thermophila, and Methanosarcina barkeri MS and Fusaro, regardless of the substrate used for growth. Structural analysis of the lipid moiety freed of polar head groups revealed that the hydroxydiether lipids of all the Methanosarcina strains were hydroxylated at position 3 of sn-2 phytanyl chains. The finding that Methanosarcina strains synthesize the same hydroxydiether structure suggests that this is a taxonomic characteristic of the genus. Methanococcus voltae produced minor amounts of the 3-hydroxydiether characteristic of Methanosarcina spp. and also the 3′-hydroxydiether described previously for Methanosaeta concilii.

51 citations

Journal ArticleDOI
TL;DR: Two signals were seen in thionin-oxidized enzyme, one with a line shape suggestive of Cu(II), and the other resembling that of a [3Fe-4S] cluster, and the enzymes nonphysiological substrate, CO, caused several spectral changes to the reduced enzyme.

51 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
20237
202212
202112
202012
20197
201818