mesna and 7-mercaptoheptanoylthreonine-phosphate

Methanogenesis, the anaerobic production of methane from CO2 or simple carbon compounds, requires seven organic coenzymes. This review describes pathways for the biosynthesis of methanofuran, 5,6,7,8-tetrahydromethanopterin, coenzyme F420, coenzyme M (2-mercaptoethanesulfonic acid) and coenzyme B (7-mercaptoheptanoyl-L-threonine phosphate). Spectroscopic evidence for the pathways is reviewed and recent efforts are described to identify and characterize the biosynthetic enzymes from methanogenic archaea. The literature from 1971 to September 2001 is reviewed, and 169 references are cited.

Topics: Archaea; Carbon Dioxide; Coenzymes; Diterpenes; Euryarchaeota; Furans; Genomics; Magnetic Resonance Spectroscopy; Mesna; Methane; Molecular Structure; Phosphothreonine; Pterins; Riboflavin

Our current knowledge of the pathways and genes involved in the biosynthesis of the methanogenic coenzymes methanopterin, coenzyme B, methanofuran, coenzyme F420, and coenzyme M is presented. Proposed reaction mechanisms for several of the novel reactions involved in the pathways are presented.

Topics: Coenzymes; Furans; Gene Expression Regulation, Archaeal; Mesna; Methanococcus; Molecular Structure; Phosphothreonine; Pterins; Riboflavin

Glutathione metabolism is associated with oxygenic cyanobacteria and the oxygen-utilizing purple bacteria, but is absent in many other prokaryotes. This review focuses on novel thiols found in those bacteria lacking glutathione. Included are glutathione amide and its perthiol, produced by phototrophic purple sulfur bacteria and apparently involved in their sulfide metabolism. Among archaebacteria, coenzyme M (2-mercaptoethanesulfonic acid) and coenzyme B (7-mercaptoheptanoylthreonine phosphate) play central roles in the anaerobic production of CH4 and associated energy conversion by methanogens, whereas the major thiol in the aerobic phototrophic halobacteria is gamma-glutamylcysteine. The highly aerobic actinomycetes produce mycothiol, a conjugate of N-acetylcysteine with a pseudodisaccharide of glucosamine and myo-inositol, AcCys-GlcNalpha(1 --> 1)Ins, which appears to play an antioxidant role similar to glutathione. Ergothioneine, also produced by actinomycetes, remains a mystery despite many years of study. Available data on the biosynthesis and metabolism of these and other novel thiols is summarized and key areas for additional study are identified.

Topics: Cysteine; Disaccharides; Ergothioneine; Glutathione; Glycopeptides; Inositol; Mesna; Models, Chemical; Phosphothreonine; Prokaryotic Cells; Pyrazoles; Sulfhydryl Compounds

Topics: Cobamides; Coenzymes; Euryarchaeota; Furans; Mesna; Metalloporphyrins; Metalloproteins; Methane; Molecular Structure; Nickel; Phosphothreonine; Pterins; Riboflavin

Find your path: Methyl-coenzyme M reductase (MCR, turquoise) reversibly catalyzes the reduction of methyl-coenzyme M (methyl-S-CoM) with coenzyme B (CoB-SH) to form methane and the heterodisulfide. Recently, spectroscopic methods were used to detect trapped intermediates in a stopped-flow system, and CoM-S-Ni

Topics: Electron Spin Resonance Spectroscopy; Mesna; Methane; Models, Molecular; Molecular Structure; Phosphothreonine

Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mM). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(Ni(I))·CH3SCoM) is highly favored (Kd = 79 μM). Only then can the chemical reaction occur (kobs = 20 s(-1) at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(Ni(II))·CoB7S(-)·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates.

Topics: Archaeal Proteins; Biocatalysis; Electron Spin Resonance Spectroscopy; Kinetics; Mesna; Methane; Methanobacteriaceae; Models, Biological; Models, Chemical; Nickel; Oxidoreductases; Phosphothreonine; Protein Binding; Protein Multimerization; Protein Subunits; Spectrometry, Fluorescence; Substrate Specificity

In methanogenic archaea growing on H(2) and CO(2) the first step in methanogenesis is the ferredoxin-dependent endergonic reduction of CO(2) with H(2) to formylmethanofuran and the last step is the exergonic reduction of the heterodisulfide CoM-S-S-CoB with H(2) to coenzyme M (CoM-SH) and coenzyme B (CoB-SH). We recently proposed that in hydrogenotrophic methanogens the two reactions are energetically coupled via the cytoplasmic MvhADG/HdrABC complex. It is reported here that the purified complex from Methanothermobacter marburgensis catalyzes the CoM-S-S-CoB-dependent reduction of ferredoxin with H(2). Per mole CoM-S-S-CoB added, 1 mol of ferredoxin (Fd) was reduced, indicating an electron bifurcation coupling mechanism: 2H(2) + Fd(OX) + CoM-S-S-CoB-->Fd(red)(2-) + CoM-SH + CoB-SH + 2H(+). This stoichiometry of coupling is consistent with an ATP gain per mole methane from 4 H(2) and CO(2) of near 0.5 deduced from an H(2)-threshold concentration of 8 Pa and a growth yield of up to 3 g/mol methane.

Topics: Carbon Dioxide; Chromatography, High Pressure Liquid; Disulfides; Ferredoxins; Hydrogen; Magnetic Resonance Spectroscopy; Mesna; Methane; Methanobacteriaceae; Metronidazole; Oxidation-Reduction; Phosphothreonine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Methyl-coenzyme M reductase catalyzes the reversible synthesis of methane from methyl-coenzyme M in methanogenic and ANME-1 and ANME-2 Archaea. The purification procedure for methyl-coenzyme M reductase from Methanothermobacter marburgensis is described. The procedure is an accumulation of almost 30 years of research on MCR starting with the first purification described by Ellefson and Wolfe (Ellefson, W.L., and Wolfe, R.S. (1981). Component C of the methylreductase system of Methanobacterium. J. Biol. Chem.256, 4259-4262). To provide a context for this procedure, some background information is provided, including a description of whole cell experiments that provided much of our knowledge of the behavior and properties of methyl-coenzyme M reductase.

Topics: Mesna; Methanobacteriaceae; Oxidoreductases; Phosphothreonine

Coenzyme M (CoM) and coenzyme B (CoB) are essential for methane production by the euryarchaea that employ this specialized anaerobic metabolism. Two pathways are known to produce CoM, 2-mercaptoethanesulfonate, and both converge on the 2-oxoacid sulfopyruvate. These cells have recruited the rich biochemistry of amino acid and 2-oxoacid metabolizing enzymes to produce a compound that resembles oxaloacetate, but with a more stable and acidic sulfonate group. 7-Mercaptoheptanoylthreonine phosphate, CoB, likewise owes its carbon backbone to a 2-oxoacid. Three enzymes recruited from leucine biosynthesis have evolved to catalyze the elongation of 2-oxoglutarate to 2-oxosuberate in CoB biosynthesis. This chapter describes the enzymology, synthesis, and analytical techniques used to study 2-oxoacid metabolism in these pathways. Protein structure and mechanistic information from enzymes provide insight into the evolution of new enzymatic activity, and the evolution of substrate specificity from promiscuous enzyme scaffolds.

Topics: Euryarchaeota; Keto Acids; Mesna; Methane; Phosphothreonine

Acetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H₂ as an intermediate, although the great majority of acetotrophs are unable to metabolize H₂. The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf (Rhodobacter nitrogen fixation) complexes distributed in the domain Bacteria distinguishes non-H₂-utilizing Methanosarcina acetivorans from H₂-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens.. A component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H2-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans.. The results support roles for ferredoxin, cytochrome c and MP in the energy-conserving electron transport pathway of non-H₂-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.

Topics: Acetates; DNA, Bacterial; Electron Transport; Electron Transport Chain Complex Proteins; Ferredoxins; Mesna; Methanosarcina; Molecular Sequence Data; Phenazines; Phosphothreonine; Sequence Analysis, DNA

Methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis: the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyme and contains a highly reduced nickel-tetrapyrrole, coenzyme F(430). Methyl-SCoM must enter the active site prior to CoBSH, as species derived from methyl-SCoM are always observed bound to the F(430) nickel in the deepest part of the 30 A long substrate channel that leads from the protein surface to the active site. The seven-carbon mercaptoalkanoyl chain of CoBSH binds within a 16 A predominantly hydrophobic part of the channel close to F(430), with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 A. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conformational change that moves methyl-SCoM closer to the nickel promoting cleavage of the C-S bond of methyl-SCoM. In order to better understand the structural role of CoBSH early in the MCR mechanism, we have determined crystal structures of MCR in complex with four different CoBSH analogues: pentanoyl, hexanoyl, octanoyl, and nonanoyl derivatives of CoBSH (CoB(5)SH, CoB(6)SH, CoB(8)SH, and CoB(9)SH, respectively). The data presented here reveal that the shorter CoB(5)SH mercaptoalkanoyl chain overlays with that of CoBSH but terminates two units short of the CoBSH thiolate position. In contrast, the mercaptoalkanoyl chain of CoB(6)SH adopts a different conformation, such that its thiolate is coincident with the position of the CoBSH thiolate. This is consistent with the observation that CoB(6)SH is a slow substrate. A labile water in the substrate channel was found to be a sensitive indicator for the presence of CoBSH and HSCoM. The longer CoB(8)SH and CoB(9)SH analogues can be accommodated in the active site through exclusion of this water. These analogues react with Ni(III)-methyl, a proposed MCR catalytic intermediate of methanogenesis. The CoB(8)SH thiolate is 2.6 A closer to the nickel than that of CoBSH, but the additional carbon of CoB(9)SH only decreases the nickel thiolate distance a further 0.3 A. Although the analogues do not induce any structural changes in the substrate channel, the thiolates appear to preferentially bind at two distinct positions in the channel, one being the previously observed CoBSH thiolate position and the other being

Topics: Catalysis; Catalytic Domain; Crystallography, X-Ray; Mesna; Methane; Methanobacteriaceae; Models, Molecular; Oxidoreductases; Phosphothreonine; Protein Conformation

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the terminal step in methanogenesis using coenzyme B (CoBSH) as the two-electron donor to reduce methyl-coenzyme M (methyl-SCoM) to form methane and the heterodisulfide, CoBS-SCoM. The active site of MCR contains an essential redox-active nickel tetrapyrrole cofactor, coenzyme F(430), which is active in the Ni(I) state (MCR(red1)). Several catalytic mechanisms have been proposed for methane synthesis that mainly differ in whether an organometallic methyl-Ni(III) or a methyl radical is the first catalytic intermediate. A mechanism was recently proposed in which methyl-Ni(III) undergoes homolysis to generate a methyl radical (Li, X., Telser, J., Kunz, R. C., Hoffman, B. M., Gerfen, G., and Ragsdale, S. W. (2010) Biochemistry 49, 6866-6876). Discrimination among these mechanisms requires identification of the proposed intermediates, none of which have been observed with native substrates. Apparently, intermediates form and decay too rapidly to accumulate to detectible amounts during the reaction between methyl-SCoM and CoBSH. Here, we describe the reaction of methyl-SCoM with a substrate analogue (CoB(6)SH) in which the seven-carbon heptanoyl moiety of CoBSH has been replaced with a hexanoyl group. When MCR(red1) is reacted with methyl-SCoM and CoB(6)SH, methanogenesis occurs 1000-fold more slowly than with CoBSH. By transient kinetic methods, we observe decay of the active Ni(I) state coupled to formation and subsequent decay of alkyl-Ni(III) and organic radical intermediates at catalytically competent rates. The kinetic data also revealed substrate-triggered conformational changes in active Ni(I)-MCR(red1). Electron paramagnetic resonance (EPR) studies coupled with isotope labeling experiments demonstrate that the radical intermediate is not tyrosine-based. These observations provide support for a mechanism for MCR that involves methyl-Ni(III) and an organic radical as catalytic intermediates. Thus, the present study provides important mechanistic insights into the mechanism of this key enzyme that is central to biological methane formation.

Topics: Acetylation; Electron Spin Resonance Spectroscopy; Kinetics; Mesna; Methane; Methanobacteriaceae; Oxidoreductases; Phosphothreonine; Spectrophotometry; Tyrosine

Methyl-coenzyme M reductase (MCR) catalyses the formation of methane from methyl-coenzyme M (CH(3)-S-CoM) and coenzyme B (HS-CoB) in methanogenic archaea. The enzyme has an alpha(2)beta(2)gamma(2) subunit structure forming two structurally interlinked active sites each with a molecule F(430) as a prosthetic group. The nickel porphinoid must be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-based electron paramagnetic resonance (EPR) signal and a UV-vis spectrum with an absorption maximum at 385 nm. This state is called the MCR-red1 state. In the presence of coenzyme M (HS-CoM) and coenzyme B the MCR-red1 state is in part converted reversibly into the MCR-red2 state, which shows a rhombic Ni(I)-based EPR signal and a UV-vis spectrum with an absorption maximum at 420 nm. We report here for MCR from Methanothermobacter marburgensis that the MCR-red2 state is also induced by several coenzyme B analogues and that the degree of induction by coenzyme B is temperature-dependent. When the temperature was lowered below 20 degrees C the percentage of MCR in the red2 state decreased and that in the red1 state increased. These changes with temperature were fully reversible. It was found that at most 50% of the enzyme was converted to the MCR-red2 state under all experimental conditions. These findings indicate that in the presence of both coenzyme M and coenzyme B only one of the two active sites of MCR can be in the red2 state (half-of-the-sites reactivity). On the basis of this interpretation a two-stroke engine mechanism for MCR is proposed.

Topics: Binding Sites; Electron Spin Resonance Spectroscopy; Kinetics; Mesna; Methanobacteriaceae; Oxidation-Reduction; Oxidoreductases; Phosphothreonine; Temperature

The synthesis of formyl-methanofuran and the reduction of the heterodisulfide (CoM-S-S-CoB) of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) are two crucial, H2-dependent reactions in the energy metabolism of methanogenic archaea. The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures ( pH2). Formyl-methanofuran synthesis is an endergonic reaction (DeltaG degrees ' = +16 kJ.mol-1). By analyzing the concentration ratios between formyl-methanofuran and methanofuran in the cells, free energy changes under experimental conditions (DeltaG') were found to range between +10 and +35 kJ.mol-1 depending on the pH2 applied. The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions. Heterodisulfide reduction (DeltaG degrees ' = -40 kJ.mol-1) was associated with free energy changes as high as -55 to -80 kJ.mol-1. The values were determined by analyzing the concentrations of CoM-S-S-CoB, HS-CoM and HS-CoB in methane-forming cells operating under a variety of hydrogen partial pressures. Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction. Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at pH2<0.12 bar, whilst the number was four for cells grown above that concentration. The shift occurred within a narrow pH2 span around 0.12 bar. The findings suggest that the methanogens regulate the bioenergetic machinery involved in CoM-S-S-CoB reduction and proton pumping in response to the environmental hydrogen concentrations.

Topics: Aldehyde Oxidoreductases; Archaeal Proteins; Cell Division; Disulfides; Energy Metabolism; Furans; Hydrogen-Ion Concentration; Mesna; Methanobacteriaceae; Microbiological Techniques; Oxidoreductases; Phosphothreonine; Protons; Sodium

Methyl-coenzyme M reductase (MCR) catalyzes the reaction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. At the active site, it contains the nickel porphinoid F430, which has to be in the Ni(I) oxidation state for the enzyme to be active. How the substrates interact with the active site Ni(I) has remained elusive. We report here that coenzyme M (HS-CoM), which is a reversible competitive inhibitor to methyl-coenzyme M, interacts with its thiol group with the Ni(I) and that for interaction the simultaneous presence of coenzyme B is required. The evidence is based on X-band continuous wave EPR and Q-band hyperfine sublevel correlation spectroscopy of MCR in the red2 state induced with 33S-labeled coenzyme M and unlabeled coenzyme B.

Topics: Electron Spin Resonance Spectroscopy; Mesna; Metalloporphyrins; Nickel; Oxidoreductases; Phosphothreonine; Sulfhydryl Compounds

Hybrid density functional theory has been used to investigate the catalytic mechanism of methyl-coenzyme M reductase (MCR), an essential enzyme in methanogenesis. In a previous study of methane formation, a scheme was suggested involving oxidation of Ni(I) in the starting square-planar coordination to the high-spin Ni(II) form in the CoM-S-Ni(II)F(430) octahedral intermediate. The methyl radical, concomitantly released by methyl-coenzyme M (CoM), is rapidly quenched by hydrogen atom transfer from the coenzyme B (CoB) thiol group, yielding methane as the first product of the reaction. The present investigation primarily concerns the second and final step of the reaction: oxidation of CoB and CoM to the CoB-S-S-CoM heterodisulfide product and reduction of nickel back to the Ni(I) square-planar form. The activation energy for the second step is found to be around 10 kcal/mol, implying that the first step of methane formation with an activation energy of 20 kcal/mol should be rate-limiting. An oxygen of the Gln147 residue, occupying the rear axial position in the oxidized Ni(II) state, is shown to stabilize the intermediate by 6 kcal/mol, thereby slightly decreasing the barrier for the preceding rate-limiting transition state. The mechanism suggested is discussed in the context of available experimental data. An analysis of the flexibility of the F(430) cofactor during the reaction cycle is also given.

Topics: Catalysis; Mesna; Metalloporphyrins; Methane; Models, Chemical; Models, Molecular; Nickel; Oxidation-Reduction; Oxidoreductases; Phosphothreonine; Sulfides

Heterodisulfide reductases (HDRs) from methanogenic archaea are iron-sulfur flavoproteins or hemoproteins that catalyze the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). In this work, the ground- and excited-state electronic properties of the paramagnetic Fe-S clusters in Methanothermobacter marburgensis HDR have been characterized using the combination of electron paramagnetic resonance and variable-temperature magnetic circular dichroism spectroscopies. The results confirm multiple S=1/2 [4Fe-4S](+) clusters in dithionite-reduced HDR and reveal spectroscopically distinct S=1/2 [4Fe-4S](3+) clusters in oxidized HDR samples treated separately with the CoM-SH and CoB-SH cosubstrates. The active site of HDR is therefore shown to contain a [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. The catalytic mechanism of HDR is discussed in light of the crystallographic and spectroscopic studies of the related chloroplast ferredoxin:thioredoxin reductase class of disulfide reductases.

Topics: Archaeal Proteins; Catalytic Domain; Disulfides; Iron; Iron-Sulfur Proteins; Mesna; Methanobacteriaceae; Models, Chemical; Oxidoreductases; Phosphothreonine; Sulfur

Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from Methanosarcina thermophila contains two b-hemes and two [Fe(4)S(4)] clusters. The physiological electron donor for HDR appears to be methanophenazine (MPhen), a membrane-bound cofactor, which can be replaced by a water-soluble analog, 2-hydroxyphenazine (HPhen). This report describes the electron transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degrees C, K(m) (HPhenH(2)) = 92 microm, K(m) (CoB-S-S-CoM) = 144 microm. Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using CO and diphenylene iodonium indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH(2). Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH(2), suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH(2). We propose the following electron transfer pathway: HPhenH(2) to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM.

Topics: Carbon Monoxide; Electron Spin Resonance Spectroscopy; Electron Transport; Flow Injection Analysis; Iron-Sulfur Proteins; Mersalyl; Mesna; Methanosarcina; Onium Compounds; Oxidoreductases; Phenazines; Phosphothreonine

The biochemical mechanism for the formation of the amide bond in N-(7-mercaptoheptanoyl)-L-threonine phosphate (HS-HTP) has been studied by measuring the incorporation of L-[3-(3)H]threonine into N-(7-mercaptoheptanoyl)-L-threonine (HS-HT) by cell extracts (CE) of Methanosarcina thermophila incubated with different precursors. Synthesis of HS-HT was observed from L-[3-(3)H]threonine and 7-mercaptoheptanoic acid (HS-H) when the incubations were conducted with either crude CE or Sephadex column-purified CE. In the presence of CE, the synthesis of HS-HT was found to be inhibited 66% by preincubation of the extract with ATPase, indicating that ATP was involved in the biosynthesis. In spite of this indication of ATP involvement in the coupling reaction, incubation of the crude CE with L-[3-(3)H]threonine, HS-H, and ATP was found to inhibit the formation of HS-HT. In contrast, the synthesis of HS-HT in the presence of Sephadex column-purified CE was found to be stimulated by the addition of ATP. Incubation of the crude CE with the CoA thioester of 7-mercaptoheptanoic acid (HS-HCoA) or the mixed disulfide formed between coenzyme M and 7-mercaptoheptanoic acid did not stimulate the biosynthesis. The biosynthesis of HS-HT was found to be strongly inhibited by an ethanol extract of the crude CE. This inhibition was found to be attributed to the HS-HTP present in the extract. Stimulation of HS-HT biosynthesis 300-fold was observed when the Sephadex column-purified CE was incubated with L-[3-(3)H]threonine and 7-mercaptoheptanoyl phosphate (HS-H-P). Data indicate that HS-HT is produced by the phosphorylation of HS-H to HS-H-P with ATP, which then reacts with L-threonine to produce HS-HT.

Topics: Adenosine Triphosphate; Amides; Coenzymes; Heptanoic Acids; Mesna; Methanosarcina; Phosphorylation; Phosphothreonine; Sulfhydryl Compounds; Threonine

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

Topics: Binding Sites; Catalysis; Coenzymes; Crystallography, X-Ray; Disulfides; Hydrogen; Hydrogen Bonding; Ligands; Mesna; Metalloporphyrins; Methane; Methanobacterium; Models, Molecular; Nickel; Oxidation-Reduction; Oxidoreductases; Phosphothreonine; Protein Conformation; Protein Folding; Protein Structure, Secondary

Topics: Carbon Dioxide; Coenzymes; Energy Metabolism; Euryarchaeota; Mesna; Methane; Nickel; Oxidoreductases; Phosphothreonine; Protein Conformation

Topics: Acetates; Carbon Dioxide; Coenzymes; Crystallography, X-Ray; Euryarchaeota; Formates; Hydrogen; Mesna; Metalloporphyrins; Methane; Methanobacterium; Oxidation-Reduction; Oxidoreductases; Phosphothreonine; Protein Conformation

During the methanogenic fermentation of acetate by Methanosarcina thermophila, the CO dehydrogenase complex cleaves acetyl coenzyme A and oxidizes the carbonyl group (or CO) to CO2, followed by electron transfer to coenzyme M (CoM)-S-S-coenzyme B (CoB) and reduction of this heterodisulfide to HS-CoM and HS-CoB (A. P. Clements, R. H. White, and J. G. Ferry, Arch. Microbiol. 159:296-300, 1993). The majority of heterodisulfide reductase activity was present in the soluble protein fraction after French pressure cell lysis. A CO:CoM-S-S-CoB oxidoreductase system from acetate-grown cells was reconstituted with purified CO dehydrogenase enzyme complex, ferredoxin, membranes, and partially purified heterodisulfide reductase. Coenzyme F420 (F420) was not required, and CO:F420 oxidoreductase activity was not detected in cell extracts. The membranes contained cytochrome b that was reduced with CO and oxidized with CoM-S-S-CoB. The results suggest that a novel CoM-S-S-CoB reducing system operates during acetate conversion to CH4 and CO2. In this system, ferredoxin transfers electrons from the CO dehydrogenase complex to membrane-bound electron carriers, including cytochrome b, that are required for electron transfer to the heterodisulfide reductase. The cytochrome b was purified from solubilized membrane proteins in a complex with six other polypeptides. The cytochrome was not reduced when the complex was incubated with H2 or CO, and H2 uptake hydrogenase activity was not detected; however, the addition of CO dehydrogenase enzyme complex and ferredoxin enabled the CO-dependent reduction of cytochrome b.

Topics: Acetates; Aldehyde Oxidoreductases; Carbon Monoxide; Cell-Free System; Cytochrome b Group; Electron Transport; Ferredoxins; Membranes; Mesna; Methanosarcina; Multienzyme Complexes; Oxidation-Reduction; Oxidoreductases; Phosphothreonine; Spectrophotometry

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.

Topics: Acetamides; Adenosine Triphosphatases; Adenosine Triphosphate; Anti-Bacterial Agents; Antifungal Agents; Hydrogen-Ion Concentration; Macrolides; Membrane Potentials; Mesna; Methanosarcina; Methyltransferases; Nitriles; Phosphothreonine; Protons; Sodium

Methyl-coenzyme M reductase (MCR) catalyses the methane-forming step in the energy metabolism of methanogenic Archaea. It brings about the reduction of methyl-coenzyme M (CH3-S-CoM) by 7-mercaptoheptanoylthreonine phosphate (H-S-HTP). Methanobacterium thermoautotrophicum contains two isoenzymes of MCR, designated MCR I and MCR II, which are expressed differentially under different conditions of growth. These two isoenzymes have been separated, purified and their catalytic and spectroscopic properties determined. Initial-velocity measurements of the two-substrate reaction showed that the kinetic mechanism for both isoenzymes involved ternary-complex formation. Double reciprocal plots of initial rates versus the concentration of either one of the two substrates at different constant concentrations of the other substrate were linear and intersected on the abcissa to the left of the 1/v axis. The two purified isoenzymes differed in their Km values for H-S-HTP and for CH3-S-CoM and in Vmax. MCR I displayed a Km for H-S-HTP of 0.1-0.3 mM, a Km for CH3-S-CoM of 0.6-0.8 mM and a Vmax of about 6 mumol.min-1 x mg-1 (most active preparation). MCR II showed a Km for H-S-HTP of 0.4-0.6 mM, a Km for CH3-S-CoM of 1.3-1.5 mM and a Vmax of about 21 mumol.min-1 x mg-1 (most active preparation). The pH optimum of MCR I was 7.0-7.5 and that of MCR II 7.5-8.0. Both isoenzymes exhibited very similar temperature activity optima and EPR properties. The location of MCR I and of MCR II within the cell, determined via immunogold labeling, was found to be essentially identical. The possible basis for the existence of MCR isoenzymes in M. thermoautotrophicum is discussed.

Topics: Catalysis; Electron Spin Resonance Spectroscopy; Immunohistochemistry; Isoenzymes; Kinetics; Mesna; Methane; Methanobacterium; Oxidation-Reduction; Oxidoreductases; Phosphothreonine; Spectrophotometry, Ultraviolet

Purified S-methyl-coenzyme M reductase (methylreductase) exhibits a very low fraction of its in vivo activity, suggesting either enzyme inactivation during cell lysis and chromatographic purification or the lack of an activating component in assay mixtures. Evidence that all methylreductase molecules in the purified protein can catalyze slow substrate turnover is found in a study of turnover-dependent in vitro incorporation of radiolabeled HS-CoM at the enzyme active site (Hartzell, P. L., Donnelly, M. I., and Wolfe, R. S. (1987) J. Biol. Chem. 262, 5581-5586). We have conducted active site titrations of purified methylreductase and of a highly active partially purified preparation (Rospert, S., Bocher, R., Albracht, S. P. J., and Thauer, R. K. (1991) FEBS Lett. 291, 371-375) using the reversible competitive inhibitor bromopropanesulfonate (K(i) = 0.05 microM). Curve fitting the data based on an equilibrium binding model shows that 0.1-1.4% of purified methylreductase has functional inhibitor binding sites while up to 25% of a highly active preparation binds the inhibitor. An EPR titration of highly active methylreductase with this inhibitor is consistent with this result, showing that the MCR-red1 and -red2 EPR signals (Albracht, S. P. J., Ankel-Fuchs, D., Bocher, R., Ellermann, J., Moll, J., van der Zwann, J. W., and Thauer, R. K. (1988) Biochim. Biophys. Acta 955, 86-102) are titrated in parallel with this active fraction. Attempts to observe turnover-dependent uptake of radiolabel from [thio-35S]2-methylthioethane-sulfonate by methylreductase were unsuccessful. These results suggest that the low activity of purified methylreductase is due primarily to low percentages of catalytically competent enzyme.

Topics: Alkanesulfonates; Binding Sites; Binding, Competitive; Chromatography, High Pressure Liquid; Disulfides; Dithiothreitol; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Euryarchaeota; Mesna; Oxidation-Reduction; Oxidoreductases; Phosphothreonine

Methyl-coenzyme-M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M [2-(methylthio)ethanesulfonate] and 7-mercaptoheptanoylthreonine phosphate in methanogenic archaea. The enzyme contains the nickel porphinoid coenzyme F430 as a prosthetic group. In the active, reduced (red) state, the enzyme displays two characteristic EPR signals, MCR-red1 and MCR-red2, probably derived from Ni(I). In the presence of the substrate methyl-coenzyme M, the rhombic MCR-red2 signal is quantitatively converted to the axial MCR-red1 signal. We report here on the effects of inhibitory substrate analogues on the EPR spectrum of the enzyme. 3-Bromopropanesulfonate (BrPrSO3), which is the most potent inhibitor of MCR known to date (apparent Ki = 0.05 microM), converted the EPR signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal designated MCR-BrPrSO3. 3-Fluoropropanesulfonate (apparent Ki < 50 microM) and 3-iodopropanesulfonate (apparent Ki < 1 microM) induced a signal identical to that induced by BrPrSO3 without affecting the line shape, despite the fact that the fluorine, bromine and iodine isotopes employed have nuclear spins of I = 1/2, I = 3/2 and I = 5/2, respectively. This finding suggests that MCR-BrPrSO3 is not the result of a close halogen-Ni(I) interaction. 7-Bromoheptanoylthreonine phosphate (BrHpoThrP) (apparent Ki = 5 microM), which is an inhibitory substrate analogue of 7-mercaptoheptanoylthreonine phosphate, converted the signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal, MCR-BrHpoThrP, similar but not identical to MCR-BrPrSO3. The results indicate that inhibition of MCR by the halogenated substrate analogues investigated above is not via oxidation of Ni(I)F430. The different MCR EPR signals are assigned to different enzyme/substrate and enzyme/inhibitor complexes.

Topics: Alkanesulfonates; Catalysis; Electron Spin Resonance Spectroscopy; Mesna; Methane; Methanobacterium; Nickel; Oxidoreductases; Phosphothreonine; Substrate Specificity

Methanosarcina strain Gö1 was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (c547 and c552) cytochromes in membranes from Methanosarcina strain Gö1. The midpoint potentials determined were Em,7 = -135 +/- 5 and -240 +/- 11 mV (b-type cytochromes) and Em,7 = -140 +/- 10 and -230 +/- 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 mumol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide.

Topics: Cytochromes; Electron Transport; Mesna; Methanol; Methanosarcina; Oxidation-Reduction; Phosphothreonine; Potentiometry; Riboflavin; Spectrum Analysis; Temperature

The 2-(methylthio)ethanesulfonic acid (CH3-S-CoM) reductase catalyzes the final methane-yielding reaction in fastidiously anaerobic methanogenic archaebacteria. This step involves the reductive demethylation of CH3-S-CoM with reducing equivalents from N-7-(mercaptoheptanoyl)-L-threonine O3-phosphate (HS-HTP) to yield methane and the nonsymmetrical disulfide of 2-mercaptoethanesulfonic acid and HS-HTP. We chemically synthesized modified analogs of CH3-S-CoM (which has two carbons in the ethylene bridge) and of HS-HTP (which has seven carbons in the side chain); analog pairs possessed an overall correct number of side chain carbons (i.e., a total of nine in combination). They were simultaneously added to anaerobic cell extracts of Methanobacterium thermoautotrophicum delta H. The ability of the extracts to reductively demethylate the modified substrates was tested by gas chromatography. We also describe here previously unknown inhibitors of methanogenesis, 6-(methylthio)hexanoyl-L-threonine O3-phosphate (a structural analog of HS-HTP) and sodium bromomethanesulfonic acid (a structural analog of CH3-S-CoM). Both analogs were found to be effective competitive inhibitors with respect to HS-HTP. These substrate analogs were also found to inhibit a recently described photoactivation of homogeneous inactive reductase (K. D. Olson, C. W. McMahon, and R. S. Wolfe, Proc. Natl. Acad. Sci. USA 88:4099-4103, 1991). In addition, we probed the mechanism of action of a potent inhibitor of the enzyme, 2-bromoethanesulfonic acid, a structural analog of CH3-S-CoM.

Topics: Binding, Competitive; Enzyme Activation; Enzyme Inhibitors; Kinetics; Light; Mesna; Methane; Methanobacterium; Oxidoreductases; Phosphothreonine; Subcellular Fractions; Substrate Specificity; Sulfides

Inactive 2-(methylthio)ethanesulfonic acid (CH3-S-CoM) reductase was partially activated by exposure to light. This simplified system replaces the complex enzymatic system of protein components A2, A3a, A3b, and ATP, which previously represented the only available means of reactivating the enzyme. Components necessary for light activation include N-(7-mercaptoheptanoyl)-L-threonine O3-phosphate (HS-HTP), CH3-S-CoM, titanium(III) citrate [Ti(III)Cit], and light above 400 nm. Photoactivation was inhibited by known inhibitors of methanogenesis: 2-bromoethanesulfonate (BES), N-(6-mercaptohexanoyl)-L-threonine O3-phosphate, N-(8-mercaptooctanoyl)-L-threonine O3-phosphate, and sodium dithionite. Methanogenesis continued when the light-activated reaction mixture was incubated in the dark. Although the specific activity was low (35 nmol of CH4 per h per mg of protein) the reaction products methane and the unsymmetrical disulfide of 2-mercaptoethanesulfonate (HS-CoM) and HS-HTP were identified. We were unable to photoactivate a reaction mixture containing the isolated prosthetic group, native F430, or its analogues.

Topics: Citrates; Citric Acid; Disulfides; Enzyme Activation; Euryarchaeota; Light; Mesna; Methane; Oxidoreductases; Phosphothreonine; Photochemistry

The reduction of the heterodisulfide of coenzyme M (H-S-CoM) and 7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80-fold to apparent homogeneity from Methanobacterium thermoautotrophicum. The native enzyme showed an apparent molecular mass of 550 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of three different subunits of apparent molecular masses 80 kDa, 36 kDa, and 21 kDa. The enzyme, which was brownish yellow, contained per mg protein 7 +/- 1 nmol FAD, 130 +/- 10 nmol non-heme iron and 130 +/- 10 nmol acid-labile sulfur, corresponding to 4 mol FAD and 72 mol FeS/mol native enzyme. The purified heterodisulfide reductase catalyzed the reduction of CoM-S-S-HTP (app. Km = 0.1 mM) with reduced benzylviologen at a specific rate of 30 mumol.min-1.mg protein-1 (kcat = 68 s-1) and the reduction of methylene blue with H-S-CoM (app. Km = 0.2 mM) plus H-S-HTP (app. Km less than 0.05 mM) at a specific rate of 15 mumol.min-1.mg-1. The enzyme was highly specific for CoM-S-S-HTP and H-S-CoM plus H-S-HTP. The physiological electron donor/acceptor remains to be identified.

Topics: Disulfides; Euryarchaeota; Mesna; Molecular Weight; Oxidation-Reduction; Oxidoreductases; Phosphothreonine; Spectrum Analysis; Substrate Specificity

An unusual fumarate reductase was purified from cell extracts of Methanobacterium thermoautotrophicum and partially characterized. Two coenzymes previously isolated from cell extracts, 2-mercaptoethane-sulfonic acid (HS-CoM) and N-(7-mercaptoheptanoyl)threonine-O3-phosphate (HS-HTP), were established as direct electron donors for fumarate reductase. By measuring the consumption of free thiol, we determined that fumarate reductase catalyzed the oxidation of HS-CoM and HS-HTP; by the direct measurement of succinate and the heterodisulfide of HS-CoM and HS-HTP (CoM-S-S-HTP), we established that these compounds were products of the fumarate reductase reaction. A number of thiol-containing compounds did not function as substrates for fumarate reductase, but this enzyme had high specific activity when HS-CoM and HS-HTP were used as electron donors. HS-CoM and HS-HTP were quantitatively oxidized by the fumarate reductase reaction, and results indicated that this reaction was irreversible. Additionally, by measuring formylmethanofuran, we demonstrated that the addition of fumarate to cell extracts activated CO2 fixation for the formation of formylmethanofuran. Results indicated that this activation resulted from the production of CoM-S-S-HTP (a compound known to be involved in the activation of formylmethanofuran synthesis) by the fumarate reductase reaction.

Topics: Disulfides; Electron Transport; Electrophoresis, Polyacrylamide Gel; Euryarchaeota; Fumarates; Furans; Mercaptoethanol; Mesna; Oxidation-Reduction; Phosphothreonine; Substrate Specificity; Succinate Dehydrogenase; Succinates; Succinic Acid; Sulfhydryl Compounds

The heterodisulfide of the two coenzymes 2-mercaptoethanesulfonic acid (coenzyme M, HS-CoM) and N-(7-mercaptoheptanoyl)threonine O3-phosphate (HS-HTP) increased the rate of CO2 reduction to methane by cell extracts 42-fold. The stimulation resulted from activation of the initial step of methanogenesis, the production of formylmethanofuran from methanofuran and CO2. These results establish a role for this heterodisulfide (CoM-S-S-HTP) in the reduction of CO2 to formylmethanofuran. Evidence indicates that CoM-S-S-HTP is the labile intermediate that accounts for the coupling of the reduction of 2-(methylthio)ethanesulfonic acid by the methylreductase to formylmethanofuran biosynthesis, the "RPG effect." The heterodisulfide was found to be labile in cell extracts due to enzyme-catalyzed reduction and possibly thioldisulfide exchange.

Topics: Carbon Dioxide; Coenzymes; Euryarchaeota; Kinetics; Mercaptoethanol; Mesna; Methane; Phosphothreonine; Threonine

The structure of component B of the methylcoenzyme M methylreductase of Methanobacterium thermoautotrophicum was recently assigned as 7-mercaptoheptanoylthreonine phosphate (HS-HTP) (Noll, K. M., Rinehart, K. L., Jr., Tanner, R.S., and Wolfe, R.S. (1986) (Proc. Natl. Acad. Sci. U.S.A. 83, 4238-4242). We report here the chemical synthesis and biochemical activity of this compound. Thiourea and 7-bromoheptanoic acid were used to to synthesize 7,7'-dithiodiheptanoic acid. This disulfide was then condensed with DL-threonine phosphate using N-hydroxysuccinimide and dicyclohexylcarbodiimide. The product was reduced with dithiothreitol to give HS-HTP. It could be oxidized in air in the presence of 2-mercaptoethanol to give the compound as it was isolated from cell extracts. The resulting product was identical to the authentic compound by 1H NMR spectroscopy, mass spectrometry, and coelution using high performance liquid chromatography. The synthetic compound is active in the in vitro methanogenic assay at concentrations comparable to the authentic compound. This confirms the structure of component B as HS-HTP and provides a means to synthesize quantities sufficient for studies of the methylreductase system.

Topics: Euryarchaeota; Indicators and Reagents; Kinetics; Mesna; Oxidoreductases; Phosphothreonine; Threonine

Purified methyl-CoM reductase of Methanobacterium thermoautotrophicum (strain Marburg) catalyzed the reduction of methyl-CoM to methane with reduced cobalamin, when either synthetic 7-mercaptoheptanoylthreonine phosphate (HS-HTP) or naturally occurring component B was present. With both compounds the same maximal specific activity was obtained and ATP was neither required nor stimulatory. These findings indicate that HS-HTP functions as component B and do not support the idea that HS-HT is only active in an adenosine monophosphorylated form.

Topics: Adenosine Triphosphate; Euryarchaeota; Kinetics; Magnesium; Mercaptoethanol; Mesna; Methane; Phosphothreonine; Threonine; Vitamin B 12

The structure of component B of the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum was recently found to be 7-mercaptoheptanoylthreonine phosphate (HS-HTP). Three potential roles for this cofactor were considered. First, a methyl thioether derivative of the cofactor was synthesized to investigate its possible role as a methyl donor. This derivative was found to be incapable of acting as a substrate for methanogenesis and proved inhibitory. Secondly, an adenylated form of the cofactor was considered as the potential active form of the coenzyme. This possibility was ruled out based upon collaborative observations with Ankel-Fuchs et al. (FEBS Lett., in press) that HS-HTP is required by the methylreductase system even when ATP is not. Finally, HS-HTP was found to act as a reductant in a partially-purified methylreductase preparation that was incubated under nitrogen. The rate of methane production from HS-HTP exceeded that from other thiols or hydrogen.

Topics: Euryarchaeota; Kinetics; Mercaptoethanol; Mesna; Methane; Oxidoreductases; Phosphothreonine; Threonine

The reduction of methyl-coenzyme M (CH3SCoM) to methane in methanogenic bacteria is dependent on component B (N-7-mercaptoheptanoyl-O-phospho-L-threonine, HSHTP). We report here that S-methyl-component B (N-7-(methylthio)heptanoyl-O-phospho-L-threonine, CH3SHTP) can substitute for neither CH3SCoM nor HSHTP in the methyl-CoM reductase reaction. Rather, CH3SHTP proved to be an inhibitor competitive with HSHTP (apparent Ki = 6 microM) and noncompetitive with CH3SCoM. These results make it very unlikely that HSHTP functions as a methyl group carrier. A role for HSHTP as direct electron donor for CH3SCoM reduction to CH4 is proposed.

Topics: Chromatography, Gas; Euryarchaeota; Mercaptoethanol; Mesna; Methane; Oxidoreductases; Phosphothreonine; Threonine

When 7-mercaptoheptanoylthreonine phosphate (HS-HTP) was used as the sole source of electrons for reductive demethylation of 2-(methylthio)-ethanesulfonic acid (CH3-S-CoM) by cell extracts of Methanobacterium thermoautotrophicum strain delta H, the heterodisulfide of coenzyme M and HS-HTP (CoM-S-S-HTP) was quantitatively produced: HS-HTP + CH3-S-CoM----CH4 + CoM-S-S-HTP. CH4 and CoM-S-S-HTP were produced stoichiometrically in a ratio of 1:1. Coenzyme M (HS-CoM) inhibited HS-HTP driven methanogenesis indicating that CH3-S-CoM rather than HS-CoM was the substrate for CoM-S-S-HTP formation.

Topics: Euryarchaeota; Mercaptoethanol; Mesna; Methane; Oxidation-Reduction; Phosphothreonine; Sulfides; Threonine

Component B, the heat-stable low-molecular-weight cofactor required for methane production by dialyzed cell-free extracts of Methanobacterium thermoautotrophicum, has been purified to homogeneity and its structure assigned. Results of low-resolution fast-atom-bombardment and field-desorption mass spectrometry indicated a molecular weight of 419, and high-resolution fast-atom-bombardment mass spectrometry agreed with the molecular formula C13H26NO8PS2. Evidence from fast-atom-bombardment and field-desorption mass spectrometry and 360-MHz 1H NMR in deuterium oxide argued that the compound was isolated as a mixed disulfide with 2-mercaptoethanol; so the proposed elemental formula of the free acid, free thiol would be C11H22NO7PS (molecular weight, 343). The proposed structure for an active form of the coenzyme is 7-mercaptoheptanoylthreonine phosphate.

Topics: Amides; Euryarchaeota; Fatty Acids; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mesna; Oxidoreductases; Phosphothreonine; Sulfhydryl Compounds; Threonine