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Uwe Deppenmeier - One of the best experts on this subject based on the ideXlab platform.
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Methanosarcina flavescens sp. nov., a Methanogenic Archaeon isolated from a full-scale anaerobic digester
International Journal of Systematic and Evolutionary Microbiology, 2016Co-Authors: Tobias Kern, Martin A. Fischer, Uwe Deppenmeier, Ruth A Schmitz, Michael RotherAbstract:A novel, strictly anaerobic, Methanogenic Archaeon, strain E03.2T, was isolated from a full-scale biogas plant in Germany. Cells were non-motile sarcina-like cocci, occurring in aggregates. Strain E03.2T grew autotrophically on H2 plus CO2, and additionally cells could utilize acetate, methanol, moni-, di- and trimethylamine as carbon and energy sources; however, growth or methanogenesis on formate was not observed. Yeast extract and vitamins stimulated growth but were not mandatory. The optimal growth temperature of strain E03.2T was approximately 45 °C; maximal growth rates were obtained at about pH 7.0 in the presence of approximately 6.8 mM NaCl. The DNA G+C content of strain E03.2T was 41.3 mol%. Phylogenetic analyses based on 16S rRNA gene and mcrA sequences placed strain E03.2T within the genus Methanosarcina. Based on 16S rRNA gene sequence similarity strain E03.2T was related to seven different species of the genus Methanosarcina, but most closely related to Methanosarcina thermophila TM-1T. Phenotypic, physiological and genomic characteristics indicated that strain E03.2T represents a novel species of the genus Methanosarcina, for which the name Methanosarcina flavescens sp. nov. is proposed. The type strain is E03.2T ( = DSM 100822T = JCM 30921T).
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electron transport during aceticlastic methanogenesis by methanosarcina acetivorans involves a sodium translocating rnf complex
FEBS Journal, 2012Co-Authors: Katharina Schlegel, Uwe Deppenmeier, Cornelia U. Welte, Volker MullerAbstract:The anaerobic Methanogenic Archaeon Methanosarcina acetivorans lives under extreme energy limitation. Methanogenesis from acetate as carried out by M. acetivorans involves an anaerobic electron transport chain with ferredoxin as electron donor and heterodisulfide as electron acceptor, and so far only the heterodisulfide reductase has been shown to translocate H+. Here, we describe a second Na+-translocating coupling site in this electron transport chain. Inside-out membrane vesicles of M. acetivorans catalyzed Na+ transport coupled to an electron transport catalyzed by the ferredoxin:heterodisulfide oxidoreductase activity. Ionophore studies revealed that Na+ transport was primary and electrogenic. A ∆rnf mutant was unable to grow on acetate and the ferredoxin:heterodisulfide oxidoreductase-coupled Na+ transport was abolished. These data are consistent with the hypothesis that the Rnf complex of M. acetivorans is an Na+-translocating coupling site and the entry point of electrons derived from reduced ferredoxin into the electron transport chain leading to the heterodisulfide.
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identification and analysis of proton translocating pyrophosphatases in the Methanogenic Archaeon methanosarcina mazei
Archaea, 2002Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Sabine Lentes, Uwe DeppenmeierAbstract:Analysis of genome sequence data from the Methanogenic Archaeon Methanosarcina mazei Go1 revealed the existence of two open reading frames encoding proton-translocating pyrophosphatases (PPases). These open reading frames are linked by a 750-bp intergenic region containing TC-rich stretches and are transcribed in opposite directions. The corresponding polypeptides are referred to as Mvp1 and Mvp2 and consist of 671 and 676 amino acids, respectively. Both enzymes represent extremely hydrophobic, integral membrane proteins with 15 predicted transmembrane segments and an overall amino acid sequence similarity of 50.1%. Multiple sequence alignments revealed that Mvp1 is closely related to eukaryotic PPases, whereas Mvp2 shows highest homologies to bacterial PPases. Northern blot experiments with RNA from methanol-grown cells harvested in the mid-log growth phase indicated that only Mvp2 was produced under these conditions. Analysis of washed membranes showed that Mvp2 had a specific activity of 0.34 U mg (protein)–1. Proton translocation experiments with inverted membrane vesicles prepared from methanol-grown cells showed that hydrolysis of 1 mol of pyrophosphate was coupled to the translocation of about 1 mol of protons across the cytoplasmic membrane. Appropriate conditions for mvp1 expression could not be determined yet. The pyrophosphatases of M. mazei Go1 represent the first examples of this enzyme class in Methanogenic archaea and may be part of their energy-conserving system. Abbreviations: DCCD, N,N′-dicyclohexylcarbodiimide; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; Δp, proton motive force.
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the f420h2 dehydrogenase from methanosarcina mazei is a redox driven proton pump closely related to nadh dehydrogenases
Journal of Biological Chemistry, 2000Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Carsten Jacobi, Andre Johann, Uwe DeppenmeierAbstract:Abstract The F420H2 dehydrogenase is part of the energy conserving electron transport system of the Methanogenic Archaeon Methanosarcina mazei Go1. Here it is shown that cofactor F420H2-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H+ translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + Pi. The gene cluster encoding the F420H2 dehydrogenase ofM. mazei Go1 comprises 12 genes that are referred to as fpoA, B, C, D,H, I, J, K,L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F420H2 dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Go1. Instead, the gene product FpoF may be responsible for F420H2 oxidation and may function as the electron input part. Thus, the F420H2 dehydrogenase from M. mazeiGo1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the Methanogenic Archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F420 and a modified output module adopted to the reduction of methanophenazine.
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the f420h2 dehydrogenase frommethanosarcina mazei is a redox driven proton pump closely related to nadh dehydrogenases
Journal of Biological Chemistry, 2000Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Carsten Jacobi, Andre Johann, Tina Ide, Uwe DeppenmeierAbstract:Abstract The F420H2 dehydrogenase is part of the energy conserving electron transport system of the Methanogenic Archaeon Methanosarcina mazei Go1. Here it is shown that cofactor F420H2-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H+ translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + Pi. The gene cluster encoding the F420H2 dehydrogenase ofM. mazei Go1 comprises 12 genes that are referred to as fpoA, B, C, D,H, I, J, K,L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F420H2 dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Go1. Instead, the gene product FpoF may be responsible for F420H2 oxidation and may function as the electron input part. Thus, the F420H2 dehydrogenase from M. mazeiGo1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the Methanogenic Archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F420 and a modified output module adopted to the reduction of methanophenazine.
Hisashi Hemmi - One of the best experts on this subject based on the ideXlab platform.
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a versatile cis prenyltransferase from methanosarcina mazei catalyzes both c and o prenylations
Journal of Biological Chemistry, 2021Co-Authors: Miyako Okada, Kohichi Emi, Hideaki Unno, Mayuko Matsumoto, Hisashi HemmiAbstract:Polyprenyl groups, products of isoprenoid metabolism, are utilized in peptidoglycan biosynthesis, protein N-glycosylation, and other processes. These groups are formed by cis-prenyltransferases, which use allylic prenyl pyrophosphates as prenyl-donors to catalyze the C-prenylation of the general acceptor substrate, isopentenyl pyrophosphate. Repetition of this reaction forms (Z,E-mixed)-polyprenyl pyrophosphates, which are converted later into glycosyl carrier lipids, such as undecaprenyl phosphate and dolichyl phosphate. MM_0014 from the Methanogenic Archaeon Methanosarcina mazei is known as a versatile cis-prenyltransferase that accepts both isopentenyl pyrophosphate and dimethylallyl pyrophosphate as acceptor substrates. To learn more about this enzyme's catalytic activity, we determined the X-ray crystal structures of MM_0014 in the presence or absence of these substrates. Surprisingly, one structure revealed a complex with O-prenylglycerol, suggesting that the enzyme catalyzed the prenylation of glycerol contained in the crystallization buffer. Further analyses confirmed that the enzyme could catalyze the O-prenylation of small alcohols, such as 2-propanol, expanding our understanding of the catalytic ability of cis-prenyltransferases.
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Reconstruction of the "Archaeal" Mevalonate Pathway from the Methanogenic Archaeon Methanosarcina mazei in Escherichia coli Cells.
Applied and environmental microbiology, 2020Co-Authors: Ryo Yoshida, Tohru Yoshimura, Hisashi HemmiAbstract:The mevalonate pathway is a well-known metabolic route that provides biosynthetic precursors for myriad isoprenoids. An unexpected variety of the pathway has been discovered from recent studies on microorganisms, mainly on archaea. The most recently discovered example, called the "archaeal" mevalonate pathway, is a modified version of the canonical eukaryotic mevalonate pathway and was elucidated in our previous study using the hyperthermophilic Archaeon Aeropyrum pernix This pathway comprises four known enzymes that can produce mevalonate 5-phosphate from acetyl coenzyme A, two recently discovered enzymes designated phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, and two more known enzymes, i.e., isopentenyl phosphate kinase and isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase. To show its wide distribution in archaea and to confirm if its enzyme configuration is identical among species, the putative genes of a lower portion of the pathway-from mevalonate to isopentenyl pyrophosphate-were isolated from the Methanogenic Archaeon Methanosarcina mazei, which is taxonomically distant from A. pernix, and were introduced into an engineered Escherichia coli strain that produces lycopene, a red carotenoid pigment. Lycopene production, as a measure of isoprenoid productivity, was enhanced when the cells were grown semianaerobically with the supplementation of mevalonolactone, which demonstrates that the archaeal pathway can function in bacterial cells to convert mevalonate into isopentenyl pyrophosphate. Gene deletion and complementation analysis using the carotenogenic E. coli strain suggests that both phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase from M. mazei are required for the enhancement of lycopene production.IMPORTANCE Two enzymes that have recently been identified from the hyperthermophilic Archaeon A. pernix as components of the archaeal mevalonate pathway do not require ATP for their reactions. This pathway, therefore, might consume less energy than other mevalonate pathways to produce precursors for isoprenoids. Thus, the pathway might be applicable to metabolic engineering and production of valuable isoprenoids that have application as pharmaceuticals. The archaeal mevalonate pathway was successfully reconstructed in E. coli cells by introducing several genes from the Methanogenic or hyperthermophilic Archaeon, which demonstrated that the pathway requires the same components even in distantly related archaeal species and can function in bacterial cells.
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a heteromeric cis prenyltransferase is responsible for the biosynthesis of glycosyl carrier lipids in methanosarcina mazei
Biochemical and Biophysical Research Communications, 2019Co-Authors: Kohichi Emi, Kitty Sompiyachoke, Miyako Okada, Hisashi HemmiAbstract:Abstract Cis-prenyltransferases are enzymes responsible for the biosynthesis of glycosyl carrier lipids, natural rubber, and some secondary metabolites. Certain organisms, including some archaeal species, possess multiple genes encoding cis-prenyltransferase homologs, and the physiological roles of these seemingly-redundant genes are often obscure. Cis-prenyltransferases usually form homomeric complexes, but recent reports have demonstrated that certain eukaryotic enzymes are heteromeric protein complexes consisting of two homologous subunits. In this study, three cis-prenyltransferase homolog proteins, MM_0014, MM_0618, and MM_1083, from the Methanogenic Archaeon Methanosarcina mazei are overexpressed in Escherichia coli and partially purified for functional characterization. Coexistence of MM_0618 and MM_1083 exhibits prenyltransferase activity, while each of them alone has almost no activity. The chain-lengths of the products of this heteromeric enzyme are in good agreement with those of glycosyl carrier lipids extracted from M. mazei, which are likely di- and tetra-hydrogenated decaprenyl phosphates, suggesting that the MM_0618/MM_1083 heteromer is involved in glycosyl carrier lipid biosynthesis. MM_0014 acts as a typical homomeric cis-prenyltransferase and produces shorter products.
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a cis prenyltransferase from methanosarcina acetivorans catalyzes both head to tail and nonhead to tail prenyl condensation
FEBS Journal, 2016Co-Authors: Takuya Ogawa, Tohru Yoshimura, Kohichi Emi, Kazushi Koga, Hisashi HemmiAbstract:Cis-prenyltransferase usually consecutively catalyzes the head-to-tail condensation reactions of isopentenyl diphosphate to allylic prenyl diphosphate in the production of (E,Z-mixed) polyprenyl diphosphate, which is the precursor of glycosyl carrier lipids. Some recently discovered homologs of the enzyme, however, catalyze the nonhead-to-tail condensation reactions between allylic prenyl diphosphates. In this study, we characterize a cis-prenyltransferase homolog from a Methanogenic Archaeon, Methanosarcina acetivorans, to obtain information on the biosynthesis of the glycosyl carrier lipids within it. This enzyme catalyzes both head-to-tail and nonhead-to-tail condensation reactions. The kinetic analysis shows that the main reaction of the enzyme is consecutive head-to-tail prenyl condensation reactions yielding polyprenyl diphosphates, while the chain lengths of the major products seem shorter than expected for the precursor of glycosyl carrier lipids. On the other hand, a subsidiary reaction of the enzyme, i.e., nonhead-to-tail condensation between dimethylallyl diphosphate and farnesyl diphosphate, gives a novel diterpenoid compound, geranyllavandulyl diphosphate.
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a novel geranylgeranyl reductase from the Methanogenic Archaeon methanosarcina acetivorans displays unique regiospecificity
FEBS Journal, 2014Co-Authors: Takuya Ogawa, Susumu Asakawa, Tohru Yoshimura, Takeshi Mori, Keisuke Isobe, Hisashi HemmiAbstract:Saturation of a prenyl group to various levels is a frequently observed modification of isoprenoids. The members of the geranylgeranyl reductase family, however, are the only known enzymes responsible for such reductive modifications in archaea. A Methanogenic Archaeon, Methanosarcina acetivorans, has proteins homologous to phytoene desaturase CrtI, which is the carotenogenic enzyme that catalyzes oxidation/isomerization of phytoene to lycopene, but their function in carotenogenesis is unlikely in a methanogen that does not produce carotenoids. In the present study, we identified one of the homologues, MA1492, as a new type of archaeal geranylgeranyl reductase that is not homologous to known geranylgeranyl reductases. The expression of MA1492 in Escherichia coli cells, which were genetically modified to produce unsaturated archaeal-type lipids, led to the production of partially saturated lipid derivatives. Furthermore, we analyzed the substrate specificity of recombinant MA1492 via in vitro assays. The LC-MS, or radio-TLC, analysis of the reaction products showed that the enzyme was definitely specific to compounds containing C20 geranylgeranyl groups and reduced only one of four double bonds in a geranylgeranyl chain. The GC-MS analysis of the product from geranylgeraniol confirmed that the reduction selectively occurred on the ω-terminal double bond. The available crystallographic structure of an orthologue enzyme may explain the reaction mechanism that achieves the substrate specificity and regiospecificity. Database Microbial Genome Database (http://mbgd.genome.ad.jp/), EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle)
Sebastian Baumer - One of the best experts on this subject based on the ideXlab platform.
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identification and analysis of proton translocating pyrophosphatases in the Methanogenic Archaeon methanosarcina mazei
Archaea, 2002Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Sabine Lentes, Uwe DeppenmeierAbstract:Analysis of genome sequence data from the Methanogenic Archaeon Methanosarcina mazei Go1 revealed the existence of two open reading frames encoding proton-translocating pyrophosphatases (PPases). These open reading frames are linked by a 750-bp intergenic region containing TC-rich stretches and are transcribed in opposite directions. The corresponding polypeptides are referred to as Mvp1 and Mvp2 and consist of 671 and 676 amino acids, respectively. Both enzymes represent extremely hydrophobic, integral membrane proteins with 15 predicted transmembrane segments and an overall amino acid sequence similarity of 50.1%. Multiple sequence alignments revealed that Mvp1 is closely related to eukaryotic PPases, whereas Mvp2 shows highest homologies to bacterial PPases. Northern blot experiments with RNA from methanol-grown cells harvested in the mid-log growth phase indicated that only Mvp2 was produced under these conditions. Analysis of washed membranes showed that Mvp2 had a specific activity of 0.34 U mg (protein)–1. Proton translocation experiments with inverted membrane vesicles prepared from methanol-grown cells showed that hydrolysis of 1 mol of pyrophosphate was coupled to the translocation of about 1 mol of protons across the cytoplasmic membrane. Appropriate conditions for mvp1 expression could not be determined yet. The pyrophosphatases of M. mazei Go1 represent the first examples of this enzyme class in Methanogenic archaea and may be part of their energy-conserving system. Abbreviations: DCCD, N,N′-dicyclohexylcarbodiimide; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; Δp, proton motive force.
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the f420h2 dehydrogenase from methanosarcina mazei is a redox driven proton pump closely related to nadh dehydrogenases
Journal of Biological Chemistry, 2000Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Carsten Jacobi, Andre Johann, Uwe DeppenmeierAbstract:Abstract The F420H2 dehydrogenase is part of the energy conserving electron transport system of the Methanogenic Archaeon Methanosarcina mazei Go1. Here it is shown that cofactor F420H2-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H+ translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + Pi. The gene cluster encoding the F420H2 dehydrogenase ofM. mazei Go1 comprises 12 genes that are referred to as fpoA, B, C, D,H, I, J, K,L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F420H2 dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Go1. Instead, the gene product FpoF may be responsible for F420H2 oxidation and may function as the electron input part. Thus, the F420H2 dehydrogenase from M. mazeiGo1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the Methanogenic Archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F420 and a modified output module adopted to the reduction of methanophenazine.
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the f420h2 dehydrogenase frommethanosarcina mazei is a redox driven proton pump closely related to nadh dehydrogenases
Journal of Biological Chemistry, 2000Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Carsten Jacobi, Andre Johann, Tina Ide, Uwe DeppenmeierAbstract:Abstract The F420H2 dehydrogenase is part of the energy conserving electron transport system of the Methanogenic Archaeon Methanosarcina mazei Go1. Here it is shown that cofactor F420H2-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H+ translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + Pi. The gene cluster encoding the F420H2 dehydrogenase ofM. mazei Go1 comprises 12 genes that are referred to as fpoA, B, C, D,H, I, J, K,L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F420H2 dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Go1. Instead, the gene product FpoF may be responsible for F420H2 oxidation and may function as the electron input part. Thus, the F420H2 dehydrogenase from M. mazeiGo1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the Methanogenic Archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F420 and a modified output module adopted to the reduction of methanophenazine.
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inhibition of membrane bound electron transport of the Methanogenic Archaeon methanosarcina mazei go1 by diphenyleneiodonium
FEBS Journal, 1999Co-Authors: Jens Brodersen, Sebastian Baumer, Gerhard Gottschalk, Hansjorg Abken, Uwe DeppenmeierAbstract:The proton translocating electron transport systems (F420H2:heterodisulfide oxidoreductase and H2:heterodisulfide oxidoreductase) of Methanosarcina mazei Go1 were inhibited by diphenyleneiodonium chloride (DPI) indicated by IC50 values of 20 nmol DPI·mg–1 protein and 45 nmol DPI·mg–1 protein, respectively. These effects are due to a complex interaction of DPI with key enzymes of the electron transport chains. It was found that 2-hydroxyphenazine-dependent reactions as catalyzed by F420-nonreducing hydrogenase, F420H2 dehydrogenase and heterodisulfide reductase were inhibited. Interestingly, the H2-dependent methylviologen reduction and the heterodisulfide reduction by reduced methylviologen as catalyzed by the hydrogenase and the heterodisulfide reductase present in washed membranes were unaffected by DPI, respectively. Analysis of the redox behavior of membrane-bound cytochromes indicated that DPI inhibited CoB-S-S-CoM-dependent oxidation of reduced cytochromes and H2-dependent cytochrome reduction. Membrane-bound and purified F420H2 dehydrogenase were inhibited by DPI irrespectively whether methylviologen + metronidazole or 2-hydroxyphenazine were used as electron acceptors. Detailed examination of 2-hydroxy-phenazine-dependent F420H2-oxidation revealed that DPI is a competitive inhibitor of the enzyme, indicated by the Km value for 2-hydroxyphenazine, which increased from 35 µm to 100 µm in the presence of DPI. As DPI and phenazines are structurally similar with respect to their planar configuration we assume that the inhibitor is able to bind to positions where interaction between phenazines and components of the electron transport systems take place. Thus, electron transfer from reduced 2-hydroxyphenazine to cytochrome b2 as part of the heterodisulfide reductase and from H2 to cytochrome b1 as subunit of the membrane-bound hydrogenase is affected in the presence of DPI. In case of the F420H2 dehydrogenase electron transport from FAD or from FeS centers to 2-hydroxyphenazine is inhibited.
Jaap S. Sinninghe Damsté - One of the best experts on this subject based on the ideXlab platform.
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A polyunsaturated irregular acyclic C25 isoprenoid in a Methanogenic Archaeon
Tetrahedron Letters, 1997Co-Authors: Jaap S. Sinninghe Damsté, Stefan Schouten, N. Herbert Van Vliet, Robert Huber, Jan A. J. GeenevasenAbstract:Abstract All trans 2,6,10,15,19-pentamethylicosa-2,6,14,18-tetraene 1 has been isolated from the Methanogenic Archaeon Methanosarcina mazei and identified by 1 H and 13 C NMR spectroscopy and mass spectrometry.
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2,6,10,15,19-Pentamethylicosenes in Methanolobus bombayensis, a marine Methanogenic Archaeon, and in Methanosarcina mazei
Organic Geochemistry, 1997Co-Authors: Stefan Schouten, Robert Huber, M.j.e.c. Van Der Maarel, Jaap S. Sinninghe DamstéAbstract:2,6,10,15,19-Pentamethylicosenes (PMEs) containing three to five double bonds have been found in the Methanogenic archaea Methanosarcina mazei (DSM 3338), a strain isolated from sewage sludge, and in Methanolobus bombayensis (OCM 438), a non-extremophilic Archaeon isolated from a marine sediment. This finding gives additional support for the use of compounds with the PME carbon skeleton as markers for Methanogenic activity in marine environments.
Gerhard Gottschalk - One of the best experts on this subject based on the ideXlab platform.
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identification of a salt induced primary transporter for glycine betaine in the methanogen methanosarcina mazei go1
Applied and Environmental Microbiology, 2002Co-Authors: M Roesler, K Pfluger, H Flach, T Lienard, Gerhard Gottschalk, Volker MullerAbstract:The salt adaptation of the Methanogenic Archaeon Methanosarcina mazei Go1 was studied at the physiological and molecular levels. The freshwater organism M. mazei Go1 was able to adapt to salt concentrations up to 1 M, and the addition of the compatible solute glycine betaine to the growth medium facilitated adaptation to higher salt concentrations. Transport studies with cell suspensions revealed a salt-induced glycine betaine uptake activity in M. mazei Go1, and inhibitor studies argue for a primary transport device. Analysis of the genome of M. mazei Go1 identified a homolog of known primary glycine betaine transporters. This gene cluster was designated Ota (osmoprotectant transporter A). Its sequence and gene organization are very similar to those of the glycine betaine transporter OpuA of Bacillus subtilis. Northern blot analysis of otaC revealed a salt-dependent transcription of this gene. Ota is the first identified salt-induced transporter for compatible solutes in Archaea.
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identification and analysis of proton translocating pyrophosphatases in the Methanogenic Archaeon methanosarcina mazei
Archaea, 2002Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Sabine Lentes, Uwe DeppenmeierAbstract:Analysis of genome sequence data from the Methanogenic Archaeon Methanosarcina mazei Go1 revealed the existence of two open reading frames encoding proton-translocating pyrophosphatases (PPases). These open reading frames are linked by a 750-bp intergenic region containing TC-rich stretches and are transcribed in opposite directions. The corresponding polypeptides are referred to as Mvp1 and Mvp2 and consist of 671 and 676 amino acids, respectively. Both enzymes represent extremely hydrophobic, integral membrane proteins with 15 predicted transmembrane segments and an overall amino acid sequence similarity of 50.1%. Multiple sequence alignments revealed that Mvp1 is closely related to eukaryotic PPases, whereas Mvp2 shows highest homologies to bacterial PPases. Northern blot experiments with RNA from methanol-grown cells harvested in the mid-log growth phase indicated that only Mvp2 was produced under these conditions. Analysis of washed membranes showed that Mvp2 had a specific activity of 0.34 U mg (protein)–1. Proton translocation experiments with inverted membrane vesicles prepared from methanol-grown cells showed that hydrolysis of 1 mol of pyrophosphate was coupled to the translocation of about 1 mol of protons across the cytoplasmic membrane. Appropriate conditions for mvp1 expression could not be determined yet. The pyrophosphatases of M. mazei Go1 represent the first examples of this enzyme class in Methanogenic archaea and may be part of their energy-conserving system. Abbreviations: DCCD, N,N′-dicyclohexylcarbodiimide; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; Δp, proton motive force.
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the f420h2 dehydrogenase from methanosarcina mazei is a redox driven proton pump closely related to nadh dehydrogenases
Journal of Biological Chemistry, 2000Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Carsten Jacobi, Andre Johann, Uwe DeppenmeierAbstract:Abstract The F420H2 dehydrogenase is part of the energy conserving electron transport system of the Methanogenic Archaeon Methanosarcina mazei Go1. Here it is shown that cofactor F420H2-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H+ translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + Pi. The gene cluster encoding the F420H2 dehydrogenase ofM. mazei Go1 comprises 12 genes that are referred to as fpoA, B, C, D,H, I, J, K,L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F420H2 dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Go1. Instead, the gene product FpoF may be responsible for F420H2 oxidation and may function as the electron input part. Thus, the F420H2 dehydrogenase from M. mazeiGo1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the Methanogenic Archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F420 and a modified output module adopted to the reduction of methanophenazine.
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the f420h2 dehydrogenase frommethanosarcina mazei is a redox driven proton pump closely related to nadh dehydrogenases
Journal of Biological Chemistry, 2000Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Carsten Jacobi, Andre Johann, Tina Ide, Uwe DeppenmeierAbstract:Abstract The F420H2 dehydrogenase is part of the energy conserving electron transport system of the Methanogenic Archaeon Methanosarcina mazei Go1. Here it is shown that cofactor F420H2-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H+ translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + Pi. The gene cluster encoding the F420H2 dehydrogenase ofM. mazei Go1 comprises 12 genes that are referred to as fpoA, B, C, D,H, I, J, K,L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F420H2 dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Go1. Instead, the gene product FpoF may be responsible for F420H2 oxidation and may function as the electron input part. Thus, the F420H2 dehydrogenase from M. mazeiGo1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the Methanogenic Archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F420 and a modified output module adopted to the reduction of methanophenazine.
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inhibition of membrane bound electron transport of the Methanogenic Archaeon methanosarcina mazei go1 by diphenyleneiodonium
FEBS Journal, 1999Co-Authors: Jens Brodersen, Sebastian Baumer, Gerhard Gottschalk, Hansjorg Abken, Uwe DeppenmeierAbstract:The proton translocating electron transport systems (F420H2:heterodisulfide oxidoreductase and H2:heterodisulfide oxidoreductase) of Methanosarcina mazei Go1 were inhibited by diphenyleneiodonium chloride (DPI) indicated by IC50 values of 20 nmol DPI·mg–1 protein and 45 nmol DPI·mg–1 protein, respectively. These effects are due to a complex interaction of DPI with key enzymes of the electron transport chains. It was found that 2-hydroxyphenazine-dependent reactions as catalyzed by F420-nonreducing hydrogenase, F420H2 dehydrogenase and heterodisulfide reductase were inhibited. Interestingly, the H2-dependent methylviologen reduction and the heterodisulfide reduction by reduced methylviologen as catalyzed by the hydrogenase and the heterodisulfide reductase present in washed membranes were unaffected by DPI, respectively. Analysis of the redox behavior of membrane-bound cytochromes indicated that DPI inhibited CoB-S-S-CoM-dependent oxidation of reduced cytochromes and H2-dependent cytochrome reduction. Membrane-bound and purified F420H2 dehydrogenase were inhibited by DPI irrespectively whether methylviologen + metronidazole or 2-hydroxyphenazine were used as electron acceptors. Detailed examination of 2-hydroxy-phenazine-dependent F420H2-oxidation revealed that DPI is a competitive inhibitor of the enzyme, indicated by the Km value for 2-hydroxyphenazine, which increased from 35 µm to 100 µm in the presence of DPI. As DPI and phenazines are structurally similar with respect to their planar configuration we assume that the inhibitor is able to bind to positions where interaction between phenazines and components of the electron transport systems take place. Thus, electron transfer from reduced 2-hydroxyphenazine to cytochrome b2 as part of the heterodisulfide reductase and from H2 to cytochrome b1 as subunit of the membrane-bound hydrogenase is affected in the presence of DPI. In case of the F420H2 dehydrogenase electron transport from FAD or from FeS centers to 2-hydroxyphenazine is inhibited.