Component A2 of the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum.

Component A2 of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum has been purified 370-fold by liquid chromatography. Homogeneity was obtained by anaerobic preparative polyacrylamide gel electrophoresis. Component A2 is a colorless, air-stable protein consisting of a single polypeptide as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The relative molecular mass of the native protein was determined by high-performance, size exclusion chromatography to be Mr 52,000; on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a value of Mr 59,000 was obtained. When cell extract was subjected to N6-ATP-agarose affinity chromatography the methylcoenzyme M methylreductase system was resolved into two fractions; one of them was component A2. This work provides a new operational definition for component A2, i.e., its characteristic chromatographic behavior on N6-ATP-agarose. However, its functional definition is its ability to reconstitute the methylreductase activity with components A1, A3, and C. Several attempts to assign a role to component A2 are reported.     

Activation of the methylreductase system from Methanobacterium bryantii by ATP.

The methylreductase of Methanobacterium bryantii required ATP for activity. There was sufficient ATP synthesis in extracts to account for the observed activity. Hexokinase inhibited the methylreductase by competing for endogenously synthesized ATP. The uncoupler, carbonyl cyanide p-trifluoromethyoxyphenyl hydrazone, inhibited only at concentrations greater than 0.5 mM, and detergents and non-halogenated membrane-permeable-ions did not inhibit. Thus, membrane proton gradients are not important in activation. In addition, maximal activation was obtained with less than 0.25 mM ATP, was inhibited by beta, gamma-imido ATP, and was strongly temperature dependent. The activated state was very unstable, having a half-life of 5 to 15 min. After gel filtration at 5 degrees C, the methylreductase retained partial activity for a short time in the absence of ATP. These observations indicate that activation involves the modification of a protein or protein-bound cofactor of the methylreductase system.     

Component A3 of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H: resolution into two components.

Component A3 of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum (strain delta H) has been resolved into two fractions. One, named component A3a, was defined as the fraction required along with components A2 and C to produce methane from 2-(methylthio)ethanesulfonate when titanium(III) citrate was used as the sole source of electrons. The second one, named component A3b, was required when H2 and 7-mercapto-N-heptanoyl-O-phospho-L-threonine were provided as the dual source of electrons. Component A3a was a large iron-sulfur protein aggregate (Mr 500,000) and is most likely involved in providing electrons at a low potential for the reductive activation of component C.     

Reductive activation of the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H.

When titanium(III) citrate was used as electron donor for the reduction of methyl coenzyme M by the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H, component A1 was no longer required. The simpler system thus obtained required components A2, A3, and C as well as catalytic amounts of ATP, vitamin B12, and the disulfide of 7-mercaptoheptanoylthreonine phosphate in addition to titanium(III) citrate. This three component enzyme system also could produce CH4 when stoichiometric amounts of 7-mercaptoheptanoylthreonine phosphate were used as a source of electrons under an H2 atmosphere. When 7-mercaptoheptanoylthreonine phosphate or H2 was used alone no CH4 was produced, indicating a dual requirement for reducing equivalents: one to activate the methylreductase system and the other to reduce methyl coenzyme M. This is the first evidence that the activation of methyl coenzyme M methylreductase is a reductive process.     

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.     

The role of 7-mercaptoheptanoylthreonine phosphate in the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum

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.     

A simplified methylcoenzyme M methylreductase assay with artificial electron donors and different preparations of component C from Methanobacterium thermoautotrophicum delta H.

Different preparations of the methylreductase were tested in a simplified methylcoenzyme M methylreductase assay with artificial electron donors under a nitrogen atmosphere. ATP and Mg2+ stimulated the reaction. Tris(2,2'-bipyridine)ruthenium (II), chromous chloride, chromous acetate, titanium III citrate, 2,8-diaminoacridine, formamidinesulfinic acid, cob(I)alamin (B12s), and dithiothreitol were tested as electron donors; the most effective donor was titanium III citrate. Methylreductase (component C) was prepared by 80% ammonium sulfate precipitation, 70% ammonium sulfate precipitation, phenyl-Sepharose chromatography, Mono Q column chromatography, DEAE-cellulose column chromatography, or tetrahydromethanopterin affinity column chromatography. Methylreductase preparations which were able to catalyze methanogenesis in the simplified reaction mixture contained contaminating proteins. Homogeneous component C obtained from a tetrahydromethanopterin affinity column was not active in the simplified assay but was active in a methylreductase assay that contained additional protein components.     

Nickel,a component of factor F 430 from Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum, growing on medium supplemented with 2 mol ⁶³NiCl₂/l, was found to take up 1.2 mol ⁶³Ni per g cells (dry weight). More than 70% of the radioisotope was incorporated into a compound, which dissociated from the protein fraction after heat treatment, was soluble in 70% acetone, and could be purified by chromatography on QAE-Sephadex A-25, Sephadex G-25, and DEAE cellulose. The purified ⁶³Ni labelled compound had an absorption spectrum and properties identical to those of factor F ₄₃₀ and is therefore considered to be identical with factor F ₄₃₀.Factor F ₄₃₀, a compound of molecular weight higher than 1000 with an absorbance maximum at 430 nm, has recently been purified from Methanobacterium thermoautotrophicum (Gunsalus and Wolfe, 1978). The structure and function of this compound are not yet known.     

ATP activation and properties of the methyl coenzyme M reductase system in Methanobacterium thermoautotrophicum.

The requirement of ATP for the methyl coenzyme M methylreductase in extracts of Methanobacterium thermoautotrophicum was found to be catalytic; for each mol of ATP added, 15 mol of methane was produced from methyl coenzyme M [2-(methylthio)ethanesulfonic acid]. Other nucleotide triphosphates partially replaced ATP in activation of the reductase. All components of the reaction were found in the supernatant fraction of cell extracts after centrifugation at 100,000 X g for 1 h; optimal reaction rates occurred at 65 degrees C, at a pH range of 5.6 to 6.0, and at concentrations of ATP and MgCl2 of 1 mM and 40 mM, respectively. Chloral hydrate, chloroform, nitrite, 2,4-dinitrophenol, and viologen dyes (compounds known to inhibit methanogenesis from a variety of substrates) were found to inhibit the conversion of methyl coenzyme M to methane. Methyl coenzyme M methylreductase was shown to be present in a variety of methanogens.     

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.     

Tetrahydromethanopterin methyltransferase, a component of the methane synthesizing complex of Methanobacterium thermoautotrophicum

A new enzyme, tetrahydromethanopterin methyltransferase, which catalyzes the transfer of methyl groups from methyl-tetrahydromethanopterin to 2-mercaptoethane-sulfonate, has been found in the methane synthesizing complex of Methanobacterium thermoautotrophicum. The enzyme is oxygen sensitive and has a well defined pH optimum at pH 6.7. There was no methyl group transfer when the enzyme was heated to 100 degrees for 5 min. The product of the forward reaction, methyl-CoM, was positively identified by TLC and high voltage paper electrophoresis. The demethylation of methyl-CoM, in the absence of methane synthesis, was dependent on the addition of H4MPT which suggests that the enzyme reaction is reversible.     

Activation of the methylreductase system from Methanobacterium bryantii by corrins.

Corrins activated the methylreductase system from Methanobacterium bryantii three- to fivefold in extracts resolved from low-molecular-weight factors. Corrins did not substitute for ATP and component B, which were also required for maximal activity. The concentration of diaquacobinamides required for one-half maximal activity was 1 microM. The concentrations of cyanocobalamin, methylcobalamin, Co alpha-(5-hydroxybenzimidazoyl)-Co beta-cyanocobamide, and 5'-deoxyadenosylcobinamide required for one-half maximal activity were between 4 and 7 microM. Deoxyadenosylcobalamin was nearly inactive. Activation was independent of thiols, coenzyme M, and ATP. Activation was also observed after partial purification of the methylreductase system by agarose column chromatography. Corrins were required in catalytic concentrations, methylcobalamin was not required, and methanogenesis was enzymatic. Corrin activation of the methylreductase is a novel effect on methanogenesis. However, the physiological significance of the corrin activation is uncertain.     

Genetic transformation system in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

A wild-type strain of Methanobacterium thermoautotrophicum Marburg was transformed by DNA from strains resistant to 5-fluorouracil. Recipient cells were grown without selection on gellan gum (GELRITE) plates with DNA. Drug-resistant cells were recovered by replica plating the resulting colonies onto drug plates. Transformation required high-molecular-weight DNA with appropriate markers and was not observed on agar or in liquid media under a variety of conditions.     

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.     

Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase - engineering a thermostable ATP independent enzyme

ABSTRACT: BACKGROUND: RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers. RESULTS: To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5' pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors. CONCLUSIONS: Mutational analysis of the heat stable RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65 deg C should reduce the constraints of RNA secondary structure in RNA ligation experiments.     

HMt, a histone-related protein from Methanobacterium thermoautotrophicum delta H.

HMt, a histone-related protein, has been isolated and characterized from Methanobacterium thermoautotrophicum delta H. HMt preparations contain two polypeptides designated HMt1 and HMt2, encoded by the hmtA and hmtB genes, respectively, that have been cloned, sequenced, and expressed in Escherichia coli. HMt1 and HMt2 are predicted to contain 68 and 67 amino acid residues, respectively, and have calculated molecular masses of 7,275 and 7,141 Da, respectively. Aligning the amino acid sequences of HMt1 and HMt2 with the sequences of HMf1 and HMf2, the subunit polypeptides of HMf, a histone-related protein from the hyperthermophile Methanothermus fervidus, revealed that 40 amino acid residues (approximately 60%) are conserved in all four polypeptides. In pairwise comparisons, these four polypeptides are 66 to 84% identical. The sequences and locations of the TATA box promoter elements and ribosome binding sites are very similar upstream of the hmtA and hmtB genes in M. thermoautotrophicum and upstream of the hmfA and hmfB genes in M. fervidus. HMt binding compacted linear pUC19 DNA molecules in vitro and therefore increased their electrophoretic mobilities through agarose gels. At protein/DNA mass ratios of < 0.2:1, HMt binding caused an increase in the overall negative superhelicity of relaxed, circular DNA molecules, but at HMt/DNA mass ratios of > 0.2:1, positive supercoils were introduced into these molecules. HMt and HMf are indistinguishable in terms of their abilities to compact and constrain DNA molecules in positive toroidal supercoils in vitro. Histone-related proteins with these properties are therefore not limited to reverse gyrase-containing hyperthermophilic species.     

Methyl-coenzyme M reductase preparations with high specific activity from H2-preincubated cells of Methanobacterium thermoautotrophicum.

The study of the nickel enzyme methyl-coenzyme M reductase from methanogenic bacteria has been hampered until now by the fact that upon cell rupture the activity of the enzyme always dropped to at best only a few percent of its in vivo activity. We describe here that when Methanobacterium thermoautotrophicum cells were preincubated with 100% H2 before disintegration methyl-coenzyme M reductase activity stayed high. The cell extracts with a specific activity of 2 U/mg protein exhibited two nickel-derived EPR signals, designated MCR-red1 and MCR-red2, previously only observed in intact cells. The enzyme was purified 10-fold to a specific activity of 20 U/mg in the presence of methyl-coenzyme M, which stabilized both the activity and the EPR signal MCR-red1. The enzyme preparation displayed an UV/Vis spectrum with an absorption maximum at 386 nm and a shoulder at 420 nm. Upon inactivation of the enzyme with O2 or CHCl3, the maximum at 386 nm and the EPR signals MCR-red1 and MCR-red2 disappeared.     

Inhibition by corrins of the ATP-dependent activation and CO2 reduction by the methylreductase system in Methanobacterium bryantii.

Corrins inhibited the ATP-dependent activation of the methylreductase system and the methyl coenzyme M-dependent reduction of CO2 in extracts of Methanobacterium bryantii resolved from low-molecular-weight factors. The concentrations of cobinamides and cobamides required for one-half of maximal inhibition of the ATP-dependent activation were between 1 and 5 microM. Cobinamides were more inhibitory at lower concentrations than cobamides. Deoxyadenosylcobalamin was not inhibitory at concentrations up to 25 microM. The inhibition of CO2 reduction was competitive with respect to CO2. The concentration of methylcobalamin required for one-half of maximal inhibition was 5 microM. Other cobamides inhibited at similar concentrations, but diaquacobinamide inhibited at lower concentrations. With respect to their affinities and specificities for corrins, inhibition of both the ATP-dependent activation and CO2 reduction closely resembled the corrin-dependent activation of the methylreductase described in similar extracts (W. B. Whitman and R. S. Wolfe, J. Bacteriol. 164:165-172, 1985). However, whether the multiple effects of corrins are due to action at a single site is unknown.     

Cyclic 2,3-diphosphoglycerate as a component of a new branch in gluconeogenesis in Methanobacterium thermoautotrophicum delta H.

A unique compound, cyclic 2,3-diphosphoglycerate (cDPG), is the major soluble carbon and phosphorus solute in Methanobacterium thermoautotrophicum delta H under optimal conditions of cell growth. It is a component of an unusual branch in gluconeogenesis in these bacteria. [U-13C]acetate pulse-[12C]acetate chase methodology was used to observe the relationship between cDPG and other metabolites (2-phosphoglycerate and 2,3-diphosphoglycerate [2-PG and 2,3-DPG, respectively]) of this branch. It was demonstrated that cells could grow exponentially under conditions in which 2-PG and 2,3-DPG, rather than cDPG, were the major solutes. While the total concentration of these three phosphorylated molecules was maintained, rapid interconversion of 13C label among them was observed. Label flow from 2-PG to 2,3-DPG to cDPG to polymer is the usual direction in this pathway in exponentially growing cells, while the reverse reactions sometimes predominate in the stationary phase. Evidence of the presence of a polymeric compound in this pathway was provided by 13C nuclear magnetic resonance (one-dimensional and two-dimensional INADEQUATE) studies of solubilized cell debris.