Methanosarcina Barkeri Research Articles

Methanol: Coenzyme M Methyltransferase from Methanosarcina Barkeri

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl-coenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated methyltransferases 1 (MT1) and 2 (MT2). Transferase MT1, which is composed of a 50-kDa subunit, MtaB, and a 27-kDa corrinoid-harbouring subunit, MtaC, has been shown recently to catalyze the methylation of free cob(I)alamin with methanol [Sauer, K., Harms, U. & Thauer, R. K. (1997) Eur. J. Biochem. 243, 670-677]. We report here that this reaction is catalyzed by subunit MtaB overproduced in Escherichia coli. MtaB also catalyzed the formation of methanol from methylcobalamin and H2O, the hydrolysis being associated with a free-energy change deltaG(o)' of approximately +7.0 kJ/mol. MtaB was found to contain 1 mol zinc, and its activity to be zinc dependent (pK(Zn2+) = 9.3). The zinc dependence of the MT2 (MtaA)-catalyzed reaction is also described (pK(Zn2+) = 9.6).

The main factors of monoamine biosensor selectivity increasing

The influence of the main factors of biosensor selectivity on monoamine determination have been studied. In the composition of new biosensors amine oxidases (AO) from different sources were used: mitochondria AO from pig and rat liver, and AO from Methanosarcina barkeri 27 bacterial strain. Enzyme preparations of different purification degree, and immobilized in different ways have been studied. Potentiometric electrodes, gas-sensing electrodes, and colorimetric sensor were used as the analytical detectors in the designed biosensors. New methods for the individual determination of monoamines and for the detection of their sum in the sample have been worked out.

The iron-sulfur centers of the pyruvate:ferredoxin oxidoreductase from Methanosarcina barkeri (Fusaro)

The iron-sulfur clusters of a pyruvate:ferredoxin oxidoreductase isolated from a methanogenic archaeon, Methanosarcina barkeri (Fusaro), have been unambiguously identified for the first time. In agreement with the estimated iron and sulfur contents (Bock and Schönheit, Eur. J. Biochem., 237 (1996) 35–44), the enzyme is shown to contain three [4Fe-4S] 2+/1+ clusters, which in the reduced state give a complex EPR spectrum resulting from three distinct centres, magnetically interacting. The catalytic cycle of the enzyme was studied by visible and EPR spectroscopies. A thiamine diphosphate based radical is also an intermediate in the M. barkeri enzyme catalytic cycle. However, under anaerobic conditions, the enzyme or Clostridium pasteurianum ferredoxin iron-sulfur clusters are reduced only in the presence of both substrates, pyruvate and coenzyme A.

Kinetic data and new enzymatic activities of Methanosarcina barkeri grown on methanol as the sole carbon source

The strictly anaerobic methanogen Methanosarcina barkeri strain Fusaro (DSM 804) was fermented on a 3-liter scale in a CSTR (continuous stirred tank reactor) to obtain almost 10 g biomass dry weight per day. The generation time of the microorganism with methanol as the sole carbon source was 6.0 h, the maximal growth rate 0.115 h−1 correspondingly. In cell-free extracts from the fermented organism active hydrogenases were found. They reduced methyl viologen (MV⊕⊕), an artificial electron acceptor, molecular hydrogen being the substrate. This reaction was used to get electrochemical energy in a biochemical fuel cell. The maximum output of this fuel cell was 2.6 mW. Furthermore, cell-free extracts from M. barkeri showed alcohol dehydrogenase activity under aerobic conditions when N,N′-dimethyl-4-nitrosoaniline (NDMA) was used as an artificial electron acceptor. The NDMA-dependent alcohol dehydrogenase (NDMA-ADH) was purified to homogeneity by column chromatography. It is a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass is about 87 kDa. The enzyme is independent of free coenzyme such as NAD, NADP, FMN, FAD and F420, but it possesses a tightly but noncovalently bound NADP(H) cofactor. The purified enzyme exhibited activity only with primary alcohols including aromatic alcohols, but methanol was not accepted. It also catalyzed the stoichiometric dismutation of aldehydes, especially long-chain aldehydes, to form a half mol of each of the corresponding alcohol and acid without addition of an electron carrier. NDMA-ADH from M. barkeri is a novel type of nicotinoprotein in methanogenic bacteria.

Purification and characterization of an alcohol:N,N-dimethyl-4-nitrosoaniline oxidoreductase from the methanogen Methanosarcina barkeri DSM 804 strain Fusaro.

Cell-free extracts of Methanosarcina barkeri DSM 804 showed alcohol dehydrogenase activity under aerobic conditions when N,N-dimethyl-4-nitrosoaniline (NDMA) was used as an artificial electron acceptor. The NDMA-dependent alcohol dehydrogenase (NDMA-ADH) was purified to approximate homogeneity by column chromatography. It is most probably a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass estimated by gel filtration being about 87 kDa. The purified protein had an isoelectric point of 4.3. It possesses a tightly but noncovalently bound NADP(H) cofactor. Each subunit contains 1 mol NADP(H)/mol, about 2 mol Zn2+/mol and significant amounts of magnesium. The purified enzyme preferably oxidized primary alcohols (including benzyl alcohol). NDMA-ADH from M. barkeri also catalyzed the stoichiometric dismutation of aldehydes, especially higher aliphatic aldehydes, to form equimolar amounts of the corresponding alcohol and acid without addition of an electron carrier. The enzyme did not catalyze the dehydrogenation of methanol or the disproportionation of formaldehyde and therefore is not directly involved in methanogenesis. An alignment of the N-terminal amino acid sequence of the enzyme with the sequences of other alcohol dehydrogenases from methanogenic and nonmethanogenic bacteria indicated no significant identity. Nevertheless there was a quite interesting sequence similarity in the first 30 N-terminal amino acids to plant cinnamyl alcohol dehydrogenase. NDMA-ADH from M. barkeri is a novel type of alcohol dehydrogenase in methanogenic bacteria.

Chloroform dechlorination by a wastewater methanogenic consortium and cell extracts of Methanosarcina barkeri

Whole-cell and cell-extract assays of a wastewater methanogenic consortium and cell extracts of Methanosarcina barkeri strain MS were shown to dechlorinate chloroform (CF), predominantly to dichloromethane (DCM). The wastewater cell dechlorination reaction was supported by electron donors like methanol, acetate, butyrate, but dechlorination rates in the presence of glucose were 5-fold less. Methanogenesis in wastewater samples was almost completely inhibited (reversibly) by as little as 71 n m (8.5 ppb) aqueous CF, but DCM was only slightly inhibitory at 107 μ m (9.1 ppm) levels. The eubacterial antibiotics, ampicillin and streptomycin, inhibited approximately 40% of the dechlorination activity in the wastewater sample and the methanogenesis inhibitor bromoethanesulfonic acid (BES) inhibited 90% of the dechlorination. CF dechlorination rates were similar in cell extracts of M. barkeri (222 nmol CF/min/mg protein) and a wastewater enrichment culture (177 nmol CF/min/mg protein). BES inhibited 41% of the CF dechlorination in cell extracts of M. barkeri and 34% of the activity in the wastewater culture extracts. Coenzyme M significantly reversed the BES inhibition of both methanogenesis and CF dechlorination by whole cells of the wastewater culture, but had no effect on cell-free extract dechlorination. Boiled cell-free extracts had dechlorination rates that were 15 and 22% higher compared to unboiled rates in extracts from the wastewater culture and M. barkeri, respectively. On the basis of these studies, it is proposed that CF dechlorination in this wastewater sample is mediated by the same catalysts found in M. barkeri and that these heat-stable catalysts are functional in both the free and protein-bound forms.

Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica.

Entamoeba histolytica is an amitochondriate protozoan parasite with numerous bacterium-like fermentation enzymes including the pyruvate:ferredoxin oxidoreductase (POR), ferredoxin (FD), and alcohol dehydrogenase E (ADHE). The goal of this study was to determine whether the genes encoding these cytosolic E. histolytica fermentation enzymes might derive from a bacterium by horizontal transfer, as has previously been suggested for E. histolytica genes encoding heat shock protein 60, nicotinamide nucleotide transhydrogenase, and superoxide dismutase. In this study, the E. histolytica por gene and the adhE gene of a second amitochondriate protozoan parasite, Giardia lamblia, were sequenced, and their phylogenetic positions were estimated in relation to POR, ADHE, and FD cloned from eukaryotic and eubacterial organisms. The E. histolytica por gene encodes a 1,620-amino-acid peptide that contained conserved iron-sulfur- and thiamine pyrophosphate-binding sites. The predicted E. histolytica POR showed fewer positional identities to the POR of G. lamblia (34%) than to the POR of the enterobacterium Klebsiella pneumoniae (49%), the cyanobacterium Anabaena sp. (44%), and the protozoan Trichomonas vaginalis (46%), which targets its POR to anaerobic organelles called hydrogenosomes. Maximum-likelihood, neighbor-joining, and parsimony analyses also suggested as less likely E. histolytica POR sharing more recent common ancestry with G. lamblia POR than with POR of bacteria and the T. vaginalis hydrogenosome. The G. lamblia adhE encodes an 888-amino-acid fusion peptide with an aldehyde dehydrogenase at its amino half and an iron-dependent (class 3) ADH at its carboxy half. The predicted G. lamblia ADHE showed extensive positional identities to ADHE of Escherichia coli (49%), Clostridium acetobutylicum (44%), and E. histolytica (43%) and lesser identities to the class 3 ADH of eubacteria and yeast (19 to 36%). Phylogenetic analyses inferred a closer relationship of the E. histolytica ADHE to bacterial ADHE than to the G. lamblia ADHE. The 6-kDa FD of E. histolytica and G. lamblia were most similar to those of the archaebacterium Methanosarcina barkeri and the delta-purple bacterium Desulfovibrio desulfuricans, respectively, while the 12-kDa FD of the T. vaginalis hydrogenosome was most similar to the 12-kDa FD of gamma-purple bacterium Pseudomonas putida. E. histolytica genes (and probably G. lamblia genes) encoding fermentation enzymes therefore likely derive from bacteria by horizontal transfer, although it is not clear from which bacteria these amebic genes derive. These are the first nonorganellar fermentation enzymes of eukaryotes implicated to have derived from bacteria.

Agitation of anoxic paddy soil slurries affects the performance of the methanogenic microbial community

Rates of CH 4 production in slurries of anoxic Italian paddy soils were higher when incubated without agitation than with shaking or stirring. Stirring resulted in a drastically reduced transformation of [2- 14C]acetate to 14CH 4 and increased the relative contribution of CH 4 production from H 14CO 3 − to total methanogenesis. Numbers of acetotrophic methanogens were low (10 3 g −1 dry soil) in stirred slurries. An anoxic suspension of sterile sand which was amended with Methanosarcina barkeri and acetate produced only CH 4 if it was not stirred. In stirred anoxic paddy soil, acetate accumulated to very high concentrations (<10 mM). Propionate, butyrate and/or isopropanol also increased in stirred slurries. Hydrogen partial pressures, on the other hand, reached in all treatments a similar value of about 3–5 Pa. However, H 2 production was apparently inhibited by stirring, since H 2 accumulated only if slurries in which methanogenesis was inhibited by chloroform were not stirred. Our results indicate that measurements of metabolic rates in anoxic paddy soil are better conducted in non-agitated incubations to avoid the potential destruction of acetotrophic methanogens, syntrophic microbial associations and other microorganisms that are sensitive to mechanical forces.

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri. Purification, properties and encoding genes of the corrinoid protein MT1.

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methylcoenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated MT1 and MT2. Transferase MT1 is a corrinoid protein. The purification, catalytic properties and encoding genes of MT2 (MtaA) have been described previously [Harms, U. and Thauer, R.K. (1996) Eur. J. Biochem. 235, 653-659]. We report here on the corresponding analysis of MT1. The corrinoid protein MT1 was purified to apparent homogeneity and showed a specific activity of 750 mumol min-1 mg-1. The enzyme catalyzed the methylation of its bound corrinoid in the cob(I)amide oxidation state by methanol. In addition to this automethylation, the purified enzyme was found to catalyze the methylation of free cob(I)alamin to methylcob(III)alamin. It was composed of two different subunits designated MtaB and MtaC, with apparent molecular masses of 49 kDa and 24 kDa, respectively. The subunit MtaC was shown to harbour the corrinoid prosthetic group. The genes mtaB and mtaC were cloned and sequenced. They were found to be juxtapositioned and to form a transcription unit mtaCB. The corrinoid-harbouring subunit MtaC exhibits 35% sequence similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli.

Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri.

Reconstitution of trimethylamine-dependent coenzyme M (CoM) methylation was achieved with three purified polypeptides. Two of these polypeptides copurified as a trimethylamine methyl transfer (TMA-MT) activity detected by stimulation of the TMA:CoM methyl transfer reaction in cell extracts. The purified TMA-MT fraction stimulated the rate of methyl-CoM formation sevenfold, up to 1.7 micromol/min/mg of TMA-MT protein. The TMA-MT polypeptides had molecular masses of 52 and 26 kDa. Gel permeation of the TMA-MT fraction demonstrated that the 52-kDa polypeptide eluted with an apparent molecular mass of 280 kDa. The 26-kDa protein eluted primarily as a monomer, but some 26-kDa polypeptides also eluted with the 280-kDa peak, indicating that the two proteins weakly associate. The two polypeptides could be completely separated using gel permeation in the presence of sodium dodecyl sulfate. The corrinoid remained associated with the 26-kDa polypeptide at a molar ratio of 1.1 corrin/26-kDa polypeptide. This polypeptide was therefore designated the TMA corrinoid protein, or TCP. The TMA-MT polypeptides, when supplemented with purified methylcorrinoid:CoM methyltransferase (MT2), could effect the demethylation of TMA with the subsequent methylation of CoM and the production of dimethylamine at specific activities of up to 600 nmol/min/mg of TMA-MT protein. Neither dimethylamine nor monomethylamine served as the substrate, and the activity required Ti(III) citrate and methyl viologen. TMA-MT could interact with either isozyme of MT2 but had the greatest affinity for the A isozyme. These results suggest that TCP is uniquely involved in TMA-dependent methanogenesis, that this corrinoid protein is methylated by the substrate and demethylated by either isozyme of MT2, and that the predominant isozyme of MT2 found in TMA-grown cells is the favored participant in the TMA:CoM methyl transfer reaction.

Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri is not a flavoenzyme.

Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri (MbHdrDE) is a membrane-bound enzyme composed of a 46-kDa subunit MbHdrD and a 23-kDa subunit MbHdrE. The enzyme has been shown to contain 0.6 mol heme and 20 mol Fe/S per mol heterodimer. In addition, substoichiometric amounts of FAD, thought to be an essential component of the active enzyme, were detected. We have now obtained preparations of active heterodisulfide reductase in high yields completely devoid of a flavin. Cloning and sequencing of the genes encoding MbHdrD and MbHdrE, which were found to form a transcription unit hdrED, revealed that both subunits also lack an FAD-binding motif. MbHdr thus differs from heterodisulifde reductase from Methanobacterium thermoautotrophicum (MtHdr), which is a flavo iron-sulfur protein composed of the subunits MtHdrA (80 kDa), MtHdrB (36 kDa) and MtHdrC (21 kDa), the subunit HdrA harboring the flavin-binding site. Sequence comparisons revealed that the N-terminal third of MbHdrD, which contained two sequence motifs for [4Fe-4S] clusters, is similar to MtHdrC and that the C-terminal two thirds of MbHdrD are similar to MtHdrB. Thus, MbHdrD and MtHdrC are structurally equivalent subunits. MbHdrE shows sequence similarity to b-type cytochromes, in agreement with the finding that this subunit contains a heme. These and other results indicate that MbHdrD harbors the active site of heterodisulfide reduction and that a flavin is not involved in catalysis. Since MbHdrD contains only iron-sulfur clusters, a mechanism of disulfide reduction involving one electron rather than two electron-transfer reactions has to be considered such as operative in ferredoxin :thioredoxin reductases from chloroplasts and cyanobacteria.

Kinetic data and new enzymatic activities of methanosarcina barkeri grown on methanol as the sole carbon source

The strictly anaerobic methanogen Methanosarcina barkeri strain Fusaro (DSM 804) was fermented on a 3-liter scale in a CSTR (continuous stirred tank reactor) to obtain almost 10 g biomass dry weight per day. The generation time of the microorganism with methanol as the sole carbon source was 6.0 h, the maximal growth rate 0.115 h-1 correspondingly. In cell-free extracts from the fermented organism active hydrogenases were found. They reduced methyl viologen (MV⨁⨁), an artificial electron acceptor, molecular hydrogen being the substrate. This reaction was used to get electrochemical energy in a biochemical fuel cell. The maximum output of this fuel cell was 2.6 mW. Furthermore, cell-free extracts from M. barkeri showed alcohol dehydrogenase activity under aerobic conditions when N,N'-dimethyl-4-nitrosoaniline (NDMA) was used as an artificial electron acceptor. The NDMA-dependent alcohol dehydrogenase (NDMA-ADH) was purified to homogeneity by column chromatography. It is a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass is about 87 kDa. The enzyme is independent of free coenzyme such as NAD, NADP, FMN, FAD and F420, but it possesses a tightly but noncovalently bound NADP(H) cofactor. The purified enzyme exhibited activity only with primary alcohols including aromatic alcohols, but methanol was not accepted. It also catalyzed the stoichiometric dismutation of aldehydes, especially long-chain aldehydes, to form a half mol of each of the corresponding alcohol and acid without addition of an electron carrier. NDMA-ADH from M. barkeri is a novel type of nicotinoprotein in methanogenic bacteria.

Methanogens in paddy rice soil

The population, methanogenic activities and dominant species of methanogenic bacteria in paddy rice soils under different conditions were studied. Application of fertilizer, especially organic manure and submergence with deep water increased the population and methanogenic activities of methanogenic bacteria in rice soils. No large differences was observed among the population of methanogen in rice soils from different depths of 0-5, 5-13 and 13-18 cm. Soils, which developed from different parent material and had various use history, had notably different cell numbers and activities of methanogenic bacteria. Methanogenic activities in soils developed from fluvo-aquic soil were obviously higher than those in soils developed from quaternary red soil and coastal saline soil, and those in upland soil were pronounced lower than those in rice soil. The methanogenic bacteria that survived in air-dried rice soil could form methane after addition of water and incubation. The dominant species of methanogens were Methanobacterium formicicum, Methanobrevibacter sp., Methanosarcina mazeii and Methanosarcina barkeri.

Effect of brackish water on biogas production from cattle dung and methanogens

Semicontinuous anaerobic digestion of cattle dung using brackish waters with conductivity from 1000–15000 μS/cm was carried out in 25 l biogas plants of floating-dome design. Water with a conductivity higher than 5000 μS/cm was inhibitory to the process. Experiments using three cultures of methanogens: Methanobacterium bryantii, Methanobacterium formicicum and Methanosarcina barkeri, confirmed these findings. Methanobacterium bryantii was found to tolerate water of the highest conductivity.

Involvement of methyltransferase-activating protein and methyltransferase 2 isoenzyme II in methylamine:coenzyme M methyltransferase reactions in Methanosarcina barkeri Fusaro.

The enzyme systems involved in the methyl group transfer from methanol and from tri- and dimethylamine to 2-mercaptoethanesulfonic acid (coenzyme M) were resolved from cell extracts of Methanosarcina barkeri Fusaro grown on methanol and trimethylamine, respectively. Resolution was accomplished by ammonium sulfate fractionation, anion-exchange chromatography, and fast protein liquid chromatography. The methyl group transfer reactions from tri- and dimethylamine, as well as the monomethylamine:coenzyme M methyltransferase reaction, were strictly dependent on catalytic amounts of ATP and on a protein present in the 65% ammonium sulfate supernatant. The latter could be replaced by methyltransferase-activating protein isolated from methanol-grown cells of the organism. In addition, the tri- and dimethylamine:coenzyme M methyltransferase reactions required the presence of a methylcobalamin:coenzyme M methyltransferase (MT2), which is different from the analogous enzyme from methanol-grown M. barkeri. In this work, it is shown that the various methylamine:coenzyme M methyltransfer steps proceed in a fashion which is mechanistically similar to the methanol:coenzyme M methyl transfer, yet with the participation of specific corrinoid enzymes and a specific MT2 isoenzyme.

Activation Mechanism of Methanol:5-Hydroxybenzimidazolylcobamide Methyltransferase from Methanosarcina barkeri

Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes involved in the transmethylation reaction from methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 only binds the methyl group of methanol when the cobalt atom of its corrinoid prosthetic groups is present in the highly reduced Co(I) state. Formation of this redox state requires H2, hydrogenase, methyltransferase activation protein, and ATP. Optical and electron paramagnetic resonance spectroscopy studies were employed to determine the oxidation states and coordinating ligands of the corrinoids of MT1 during the activation process. Purified MT1 contained 1.7 corrinoids per enzyme with cobalt in the fully oxidized Co(III) state. Water and N-3 of the 5-hydroxybenzimidazolyl base served as the upper and lower ligands, respectively. Reduction to the Co(II) level was accomplished by H2 and hydrogenase. The cob(II)amide of MT1 had the base coordinated at this stage. Subsequent addition of methyltransferase activation protein and ATP resulted in the formation of base-uncoordinated Co(II) MT1. The activation mechanism is discussed within the context of a proposed model and compared to those described for other corrinoid-containing methyl group transferring proteins.

Purification and Properties of an Enzyme Involved in the ATP-dependent Activation of the Methanol:2-Mercaptoethanesulfonic Acid Methyltransferase Reaction in Methanosarcina barkeri

In Methanosarcina barkeri the transfer of the methyl group from methanol to 2-mercaptoethanesulfonic acid is catalyzed by the concerted action of two methyltransferases. The first one is the corrinoid-containing methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1), which binds the methyl group of methanol to its corrinoid prosthetic group. MT1 is only catalytically active when the cobalt atom of the corrinoid is present in the highly reduced Co(I) state. In the course of its purification and even during catalysis, MT1 becomes oxidatively inactivated. The enzyme, however, may be reductively reactivated by a suitable reducing system (hydrogen and hydrogenase), ATP, and an enzyme called methyltransferase activation protein (MAP). In order to elucidate its role in the reactivation process, MAP was purified to apparent homogeneity. The protein had an Mr = 60,000. Preincubation of the enzymic components involved with 8-azido-ATP or with ATP demonstrated MAP to be the primary site of action of ATP. In agreement herewith, the protein was autophosphorylated by [gamma-32P]ATP in a 1:1 stoichiometry. Phosphorylated MAP substituted for ATP in the activation of MT1, and the addition of increasing amounts of MAP phosphate resulted in a corresponding increase of active MT1. However, in the presence of limiting amounts of MAP, maximal activation of MT1 could be achieved during a lag phase provided ATP was present, indicating that MAP acts as a catalyst. This paper is the first to report on the presence, isolation, and function of a phosphorylated protein in a methanogenic archaeon.

The pterin lumazine inhibits growth of methanogens and methane formation

The pterin compound lumazine [2, 4-(1H, 3H)-pteridinedione] inhibited the growth of several methanogenic archaea completely at a concentration of ≤ 0.6 mM and was bacteriocidal for Methanobacterium thermoautotrophicum strain Marburg. In contrast, growth of two non-methanogenic archaea, several eubacteria, and one eukaryote was not strongly affected at much higher concentrations. In washed-cell suspensions, methanogenesis from H2 and CO2 by Mb. thermoautotrophicum or from H2 and methanol by Methanosarcina barkeri was inhibited by addition of lumazine. In cell-free extracts of Mb. thermoautotrophicum, H2-driven methane production from CO2 or CH3-S-CoM was completely inhibited by 0.6 mM lumazine. The results suggest that the compound may be useful in probing the methanogenesis pathway or in selecting against methanogens.

Methylcobamide:Coenzyme M Methyltransferase Isozymes from Methanosarcina barkeri: PHYSICOCHEMICAL CHARACTERIZATION, CLONING, SEQUENCE ANALYSIS, AND HETEROLOGOUS GENE EXPRESSION

A comparative study was made on the physicochemical characteristics of two isozymes of methylcobamide:- coenzyme M methyltransferase (MT2). Both isozymes catalyzed S-methylation of 2-thioethanesulfonate (coenzyme M) and exhibited similar apparent Km values for coenzyme M of 35 microM (MT2-A) and 20 microM (MT2-M). Weak binding to methylcobalamin was indicated by the apparent Km of 14 mM for both isozymes. Cob(I)alamin was established as the major product of the reaction, demonstrating heterolytic cleavage of the methylcobamide carbon-cobalt bond. The isozymes were shown to be zinc-containing metalloproteins. Metal ion chelators strongly inhibited both isozymes. A variety of coenzyme M analogs were tested for activity and/or inhibition. One alternative substrate 3-mercaptopropionate was discovered, with apparent Km 9 mM (MT2-A) and 10 mM (MT2-M). The results suggested an active site geometry in which coenzyme M is bound both by S-coordination to zinc, and electrostatic interaction of the sulfonate with a cationic group on the enzyme. Methanosarcina barkeri genes cmtA and cmtM encoding both isozymes were cloned and sequenced. Both genes encoded proteins with 339 amino acids and predicted molecular masses of 36-37 kDa. Active forms of both isozymes were expressed in Escherichia coli. A conserved segment with the potential for metal binding was found. The possibility of zinc involvement in catalysis of coenzyme M methylation is considered.

Catalytic properties, molecular composition and sequence alignments of pyruvate: ferredoxin oxidoreductase from the methanogenic archaeon Methanosarcina barkeri (strain Fusaro).

Methanosarcina barkeri (strain Fusaro) was grown on pyruvate as methanogenic substrate [Bock, A. K., Prieger-Kraft, A. & Schönheit, P. (1994) Arch. Microbiol. 161, 33-46]. The first enzyme of pyruvate catabolism, pyruvate oxidoreductase, which catalyzes oxidation of pyruvate to acetyl-CoA was purified about 90-fold to apparent electrophoretic homogeneity. The purified enzyme catalyzed the CoA-dependent oxidation of pyruvate with ferredoxin as an electron acceptor which defines the enzyme as a pyruvate: ferredoxin oxidoreductase. The deazaflavin, coenzyme F420, which has been proposed to be the physiological electron acceptor of pyruvate oxidoreductase in methanogens, was not reduced by the purified enzyme. In addition to ferredoxin and viologen dyes, flavin nucleotides served as electron acceptors. Pyruvate: ferredoxin oxidoreductase also catalyzed the oxidation of 2-oxobutyrate but not the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, 3-hydroxypyruvate and oxaloacetate. The apparent Km values of pyruvate:ferredoxin oxidoreductase were 70 microM for pyruvate, 6 microM for CoA and 30 microM for clostridial ferredoxin. The apparent Vmax with ferredoxin was about 30 U/mg (at 37 degrees C) with a pH optimum of approximately 7. The temperature optimum was approximately 60 degrees C and the Arrhenius activation energy was 40 kJ/mol (between 30 degrees C and 60 degrees C). The enzyme was extremely oxygen sensitive, losing 90% of its activity upon exposure to air for 1 h at 0 degrees C. Sodium nitrite inhibited the enzyme with a Ki of about 10 mM. The native enzyme had an apparent molecular mass of approximately 130 kDa and was composed of four different subunits with apparent molecular masses of 48, 30, 25, and 15 kDa which indicates that the enzyme has an alpha beta gamma delta structure. The enzyme contained 1 mol/mol thiamine diphosphate, and about 12 mol/mol each of non-heme iron and acid-labile sulfur. FAD, FMN and lipoic acid were not found. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the alpha-subunit was similar to the N-terminal amino acid sequence of the alpha-subunit of the heterotetrameric pyruvate:ferredoxin oxidoreductases of the hyperthermophiles Archaeoglobus fulgidus, Pyrococcus furiosus and Thermotoga maritima and of the mesophile Helicobacter pylori, and to the N-terminal amino acid sequence of the homodimeric pyruvate:ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the alpha-subunit of the M. barkeri enzyme and the heterodimeric pyruvate:ferredoxin oxidoreductase of the archaeon Halobacterium halobium.