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.     

Purification and characterization of the reduced-nicotinamide-dependent 2,2''-dithiodiethanesulfonate reductase from Methanobacterium thermoautotrophicum delta H.

A novel reduced nicotinamide-dependent disulfide reductase, the 2,2'-dithiodiethanesulfonate [(S-CoM)2] reductase (CoMDSR) of Methanobacterium thermoautotrophicum was purified 405-fold to electrophoretic homogeneity. Both NADPH and NADH functioned as electron donors, although rates with NADPH were three times higher. Reduced factor F420, the deazaflavin electron carrier characteristic of methanogenic bacteria, was not a substrate for the enzyme. The enzyme was most active with (S-CoM)2 but could also reduce L-cystine at 23% the (S-CoM)2 rate. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the enzyme was monomeric with an Mr of about 64,000; spectral analysis showed that it was a flavoprotein with an estimated composition of one molecule of flavin per polypeptide. Maximal activity occurred at 64 degrees C, and the pH optimum was 8.5. The apparent Km for both NADPH and (S-CoM)2 was 80 microM. The enzyme was completely inactivated by oxygen in crude cell extracts but was oxygen stable in the homogeneous state. The low activity of the CoMDSR in cell extracts as well as its relatively low rate of reducing CoM-S-S-HTP (the heterodisulfide of the two thiol cofactors involved in the last step of methanogenesis) make it unlikely that it plays a role in the methylreductase system. It may be involved in the redox balance of the cell, such as the NADPH-dependent bis-gamma-glutamylcystine reductase with which it shows physical similarity in another archaebacterium, Halobacterium halobium (A. R. Sundquist and R. C. Fahey, J. Bacteriol. 170:3459-3467, 1988). The CoMDSR might also be involved in regenerating the coenzyme M trapped as its homodisulfide, a nonutilizable form of the cofactor.     

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.     

Reductive dechlorination of 1,2-dichloroethane and chloroethane by cell suspensions of methanogenic bacteria

Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively. The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. Cells of Methanosarcina barkeri grown on H₂/CO₂ converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.Abbreviations BrES 2-bromoethanesulfonic acid - CA chloroethane - 1,2-DCA 1,2-dichloroethane - F₄₃₀ Ni(II)tetrahydro-(12, 13)-corphin with an uroporphinoid (III) ligand skeleton     

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.     

Immunocytochemical localization of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum

Cells of Methanobacterium thermoautotrophicum were fixed with glutaraldehyde, sectioned and labeled with antibodies against the subunit of component C (=methyl-CoM reductase) of methyl-CoM reductase system and with colloidal gold-labeled protein A. It was found that the gold particles were located predominantly in the vicinity of the cytoplasmic membrane, when the cells were grown under conditions where methyl-CoM reductase was not overproduced. This finding confirms the recent data obtained with Methanococcus voltae showing via the same immunocytochemical localization technique that in this organism methyl-CoM reductase is membrane associated.     

Two genetically distinct methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum strain Marburg and delta H

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaebacteria. The reductase has been characterized in detail from Methanobacterium thermoautotrophicum strain Marburg and delta H, which grow on H2 and CO2 as energy source. During purification of the enzyme we have now discovered a second methyl-coenzyme M reductase (MCR II) in the two strains, which elutes at lower salt concentration from anion-exchange columns than the enzyme (MCR I) previously characterized. MCR II is similar to MCR I in that it is also composed of three different subunits alpha, beta, and gamma but distinct from MCR I in that the gamma subunit is 5 kDa smaller, as revealed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of the alpha, beta, and gamma subunits of MCR II and MCR I were found to be different in several amino acid positions. The respective sequences showed, however, strong similarities indicating that MCR II was not derived from MCR I by limited proteolysis. The relative amounts of MCR I and MCR II present in the cells were affected by the growth conditions. When the cultures were supplied with sufficient H2 and and CO2 and the cells grew exponentially, essentially only MCR II was found. When growth was limited by the gas supply, MCR I predominated.     

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.     

Component A2 of methylcoenzyme M reductase system from Methanobacterium thermoautotrophicum delta H: nucleotide sequence and functional expression by Escherichia coli.

The gene for component A2 of the methylcoenzyme M reductase system from Methanobacterium thermoautotrophicum delta H was cloned, and its nucleotide sequence was determined. The gene for A2, designated atwA, encodes an acidic protein of 59,335 Da. Amino acid sequence analysis revealed partial homology of A2 to a number of eucaryotic and bacterial proteins in the ATP-binding cassette (ABC) family of transport systems. Component A2 possesses two ATP-binding domains. A 2.2-kb XmaI-BamHI fragment containing atwA and the surrounding open reading frames was cloned into pGEM-7Zf(+). A cell extract from this strain replaced purified A2 from M. thermoautotrophicum delta H in an in vitro methylreductase assay.     

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.     

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.     

Preparation of coenzyme M analogues and their activity in the methyl coenzyme M reductase system of Methanobacterium thermoautotrophicum.

A number of 2-(methylthio)ethanesulfonate (methyl-coenzyme M) analogues were synthesized and investigated as substrates for methyl-coenzyme M reductase, an enzyme system found in extracts of Methanobacterterium thermoautotrophicum. Replacement of the methyl moiety by an ethyl group yielded an analogue which served as a precursor for ethane formation. Propyl-coenzyme M, however, was not converted to propane. Analogues which contained additional methylene carbons such as 3-(methylthio)propanesulfonate or 4-(methylthio)butanesulfonate or analogues modified at the sulfide or sulfonate position, N-methyltaurine and 2-(methylthio)ethanol, were inactive. These analogues, in addition to a number of commercially available compounds, also were tested for their ability to inhibit the reduction of methyl-coenzyme M to methane. Bromoethanesulfonate and chloroethanesulfonate proved to be potent inhibitors of the reductase, resulting in 50% inhibition at 7.9 X 10(6) M and 7.5 X 10(5) M. Analogues to coenzyme M which contained modifications to other regions were evaluated also and found to be weak inhibitors of methane biosynthesis.     

The intrinsic DNA helicase activity of Methanobacterium thermoautotrophicum delta H minichromosome maintenance protein

Minichromosome maintenance proteins (MCMs) form a family of conserved molecules that are essential for initiation of DNA replication. All eukaryotes contain six orthologous MCM proteins that function as heteromultimeric complexes. The sequencing of the complete genomes of several archaebacteria has shown that MCM proteins are also present in archaea. The archaea Methanobacterium thermoautotrophicum contains a single MCM-related sequence. Here we report on the expression and purification of the recombinant M. thermoautotrophicum MCM protein (MtMCM) in both Escherichia coli and baculovirus-infected cells. We show that purified MtMCM protein assembles in large macromolecular complexes consistent in size with being double hexamers. We demonstrate that MtMCM contains helicase activity that preferentially uses dATP and DNA-dependent dATPase and ATPase activities. The intrinsic helicase activity of MtMCM is abolished when a conserved lysine in the helicase domain I/nucleotide binding site is mutated. MtMCM helicase unwinds DNA duplexes in a 3' --> 5' direction and can unwind up to 500 base pairs in vitro. The kinetics, processivity, and directionality of MtMCM support its role as a replicative helicase in M. thermoautotrophicum. This strongly suggests that this function is conserved for MCM proteins in eukaryotes where a replicative helicase has yet to be identified.     

Purification and properties of 5,10-methylenetetrahydromethanopterin reductase, a coenzyme F420-dependent enzyme, from Methanobacterium thermoautotrophicum strain delta H

5,10-Methylenetetrahydromethanopterin reductase was purified 22-fold to apparent homogeneity from the methanogenic bacterium Methanobacterium thermoautotrophicum. The enzyme catalyzes the reduction of 5,10-methylene- to 5-methyltetrahydromethanopterin. The electron carrier coenzyme F420 is specifically used as the cosubstrate. The reductase reaction may proceed in both directions, methylene reduction is, however, thermodynamically favored. In addition, the velocity of the reaction in this direction exceeds the reverse reaction by a factor of 26. The reductase is composed of a single subunit with an estimated Mr = 35,000. The active enzyme does not contain a flavin prosthetic group or iron-sulfur clusters, in contrast to 5,10-methylenetetrahydrofolate reductases purified from eukaryotic and eubacterial sources, which catalyze an analogous reaction as the methanogenic reductase.     

A possible new class of ribonucleotide reductase from Methanobacterium thermoautotrophicum.

The ribonucleotide reductase from the strictly anaerobic methanogen Methanobacterium thermoautotrophicum has been partially purified by ion-exchange and gel-filtration chromatography. Its molecular weight is estimated to be 100,000 by the latter step. Unlike all previously studied ribonucleotide reductases, the enzyme does not employ dithiol compounds such as dithiothreitol as artificial electron donors in in vitro assays. Inhibition of the enzyme by S-adenosylmethionine, oxygen, and azide further distinguishes it from the Escherichia coli anaerobic enzyme, the iron- and manganese-containing, and the adenosylcobalamin-dependent enzymes. Our preliminary results suggest that this enzyme has an activation mechanism different from the known classes of ribonucleotide reductases.     

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.     

Evidence for the involvement of corrinoids and factor F430 in the reductive dechlorination of 1,2-dichloroethane by Methanosarcina barkeri.

Cobalamin and the native and diepimeric forms of factor F430 catalyzed the reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethylene or chloroethane (CA) in a buffer with Ti(III) citrate as the electron donor. Ethylene was the major product in the cobalamin-catalyzed transformation, and the ratio of ethylene to CA formed was 25:1. Native F430 and 12,13-di-epi-F430 produced ethylene and CA in ratios of about 2:1 and 1:1, respectively. Cobalamin dechlorinated 1,2-DCA much faster than did factor F430. Dechlorination rates by all three catalysts showed a distinct pH dependence, correlated in a linear manner with the catalyst concentration and doubled with a temperature increase of 10 degrees C. Crude and boiled cell extracts of Methanosarcina barkeri also dechlorinated 1,2-DCA to ethylene and CA with Ti(III) citrate as the reductant. The catalytic components in boiled extracts were heat and oxygen stable and had low molecular masses. Fractionation of boiled extracts by a hydrophobic interaction column revealed that part of the dechlorinating components had a hydrophilic and part had a hydrophobic character. These chemical properties of the dechlorinating components and spectral analysis of boiled extracts indicated that corrinoids or factor F430 was responsible for the dechlorinations. The ratios of 3:1 to 7:1 of ethylene and CA formed by cell extracts suggested that both cofactors were concomitantly active.     

Purification and characterization of an anabolic fumarate reductase from Methanobacterium thermoautotrophicum.

An oxygen-sensitive fumarate reductase has been purified from the cytosol fraction of the cells of the archaebacterium Methanobacterium thermoautotrophicum. A major portion of the purification was performed inside an anaerobic chamber, employing reducing agents to maintain low redox potentials. The apparent molecular weight of the native enzyme is 78,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a minimal subunit molecular weight of about 20,000. Iodoacetamide (1 mM) and copper chloride (5 mM) caused significant loss in the enzyme activity. The optimum temperature for the enzymatic activity was 75 degrees C. The pH optimum was found to be 7.0. The fumarate reductase had an apparent Km of 0.20 mM for fumarate. Purified enzyme was colorless; spectroscopic studies indicated the absence of flavins as a cofactor. The spectral data, however, suggested the presence of an unknown cofactor tightly bound to the enzyme. Fumarate reductase is involved in the anabolic rather than the catabolic metabolism of M. thermoautotrophicum.