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[Fe]‐hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl‐tetrahydromethanopterin (methenyl‐H4MPT+) with H2. H4MPT is a C1‐carrier of methanogenic archaea. One bacterial genus, Desulfurobacterium, contains putative genes for the Hmd paralog, termed HmdII, and the HcgA–G proteins. The latter are required for the biosynthesis of the prosthetic group of Hmd, the iron–guanylylpyridinol (FeGP) cofactor. This finding is intriguing because Hmd and HmdII strictly use H4MPT derivatives that are absent in most bacteria. We identified the presence of the FeGP cofactor in D. thermolithotrophum. The bacterial HmdII reconstituted with the FeGP cofactor catalyzed the hydrogenation of derivatives of tetrahydrofolate, the bacterial C1‐carrier, albeit with low enzymatic activities. The crystal structures show how Hmd recognizes tetrahydrofolate derivatives. These findings have an impact on future biotechnology by identifying a bacterial Hmd paralog. [ABSTRACT FROM AUTHOR]

The author asserts that reactions linked to coenzyme tetrahydromethanopterin and chemical compound methanofuran, which are both involved in methanogenesis and methylotrophy, are much more widespread among both bacteria and archaea than originally thought.

[Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (Fe GP) cofactor used for H2 cleavage. The generated hydride is transferred to methenyl-tetrahydromethanopterin (methenyl-H4 MPT+). Most hydrogenotrophic methanogens contain the hmd-related genes hmd II and hmd III. Their function is still elusive. We were able to reconstitute the Hmd II holoenzyme of Methanocaldococcus jannaschii with recombinantly produced apoenzyme and the Fe GP cofactor, which is a prerequisite for in vitro functional analysis. Infrared spectroscopic and X-ray structural data clearly indicated binding of the Fe GP cofactor. Methylene-H4 MPT binding was detectable in the significantly altered infrared spectra of the Hmd II holoenzyme and in the Hmd II apoenzyme-methylene-H4 MPT complex structure. The related binding mode of the Fe GP cofactor and methenyl-H4 MPT+ compared with Hmd and their multiple contacts to the polypeptide highly suggest a biological role in Hmd II. However, holo-Hmd II did not catalyze the Hmd reaction, not even in a single turnover process, as demonstrated by kinetic measurements. The found inactivity can be rationalized by an increased contact area between the C- and N-terminal folding units in Hmd II compared with in Hmd, which impairs the catalytically necessary open-to-close transition, and by an exchange of a crucial histidine to a tyrosine. Mainly based on the presented data, a function of Hmd II as Hmd isoenzyme, H2 sensor, Fe GP-cofactor storage protein and scaffold protein for Fe GP-cofactor biosynthesis could be excluded. Inspired by the recently found binding of Hmd II to aminoacyl- tRNA synthetases and tRNA, we tentatively consider Hmd II as a regulatory protein for protein synthesis that senses the intracellular methylene-H4 MPT concentration. Database Structural data are available in the Protein Data Bank under the accession numbers ; ; and . [ABSTRACT FROM AUTHOR]

The iron–sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor. The iron is coordinated by two CO molecules, one sulphur and a pyridone derivative, which is linked via a phosphodiester bond to a guanosine base. We report here on the crystal structure of the Hmd apoenzyme from Methanocaldococcus jannaschii at 1.75Å and from Methanopyrus kandleri at 2.4Å resolution. Homodimeric Hmd reveals a unique architecture composed of one central and two identical peripheral globular units. The central unit is composed of the intertwined C-terminal segments of both subunits, forming a novel intersubunit fold. The two peripheral units consist of the N-terminal domain of each subunit. The Rossmann fold-like structure of the N-terminal domain contains a mononucleotide-binding site, which could harbour the GMP moiety of the cofactor. Another binding site for the iron-containing cofactor is most probably Cys176, which is located at the bottom of a deep intersubunit cleft and which has been shown to be essential for enzyme activity. Adjacent to the iron of the cofactor modelled as a ligand to Cys176, an extended U-shaped extra electron density, interpreted as a polyethyleneglycol fragment, suggests a binding site for the substrate methenyltetrahydromethanopterin. [Copyright &y& Elsevier]

Formylmethanofuran:tetrahydromethanopterin formyltransferase is an essential enzyme in the one-carbon metabolism of methanogenic and sulfate-reducing archaea and of methylotrophic bacteria. The enzyme, which is devoid of a prosthetic group, catalyzes the reversible formyl transfer between the two substrates coenzyme methanofuran and coenzyme tetrahydromethanopterin (H4MPT) in a ternary complex catalytic mechanism. The structure of the formyltransferase without its coenzymes has been determined earlier. We report here the structure of the enzyme in complex with both coenzymes at a resolution of 2.0Å. Methanofuran, characterized for the first time in an enzyme structure, is embedded in an elongated cleft at the homodimer interface and fixed by multiple hydrophobic interactions. In contrast, tetrahydromethanopterin is only weakly bound in a shallow and wide cleft that provides two binding sites. It is assumed that the binding of the bulky coenzymes induces conformational changes of the polypeptide in the range of 3Å that close the H4MPT binding cleft and position the reactive groups of both substrates optimally for the reaction. The key residue for substrate binding and catalysis is the strictly conserved Glu245. Glu245, embedded in a hydrophobic region and completely buried upon tetrahydromethanopterin binding, is presumably protonated prior to the reaction and is thus able to stabilize the tetrahedral oxyanion intermediate generated by the nucleophilic attack of the N5 atom of tetrahydromethanopterin onto the formyl carbon atom of formylmethanofuran. [Copyright &y& Elsevier]

The unexpected presence of archaea-like genes for tetrahydromethanopterin (H4MPT)-dependent enzymes in the completely sequenced genome of the aerobic marine planctomycetePirellulasp. strain 1 (“Rhodopirellula baltica”) and in the currently sequenced genome of the aerobic freshwater planctomyceteGemmata obscuriglobusstrain UQM2246 revives the discussion on the origin of these genes in the bacterial domain. We compared the genomic arrangement of these genes inPlanctomycetesand methylotrophic proteobacteria and performed a phylogenetic analysis of the encoded protein sequences to address the question whether the genes have been present in the common ancestor ofBacteriaandArchaeaor were transferred laterally from the archaeal to the bacterial domain and therein. Although this question could not be solved using the data presented here, some constraints on the evolution of the genes involved in archaeal and bacterial H4MPT-dependent C1-transfer may be proposed: (i) lateral gene transfer (LGT) fromArchaeato a common ancestor ofProteobacteriaandPlanctomycetesseems more likely than the presence of the genes in the common ancestor ofBacteriaandArchaea; (ii) a single event of interdomain LGT can be favored over two independent events; and (iii) the archaeal donor of the genes might have been a representative of theMethanosarcinales. In the bacterial domain, the acquired genes evolved according to distinct environmental and metabolic constraints, reflected by specific rearrangements of gene order, gene recruitment, and gene duplication, with subsequent functional specialization. During the course of evolution, genes were lost from some planctomycete genomes or replaced by orthologous genes from proteobacterial lineages. [ABSTRACT FROM AUTHOR]

Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H4SPT) rather than tetrahydrofolate (H4F) as a pterin C1 carrier. However, the genome sequence ofMethanosarcinaspecies recently revealed a cluster of genes,purN,folD,glyAandmetF, that are predicted to encode for H4F-specific enzymes. We show here forfolDandglyAfromM. barkerithat this prediction is correct: FolD (bifunctionalN5,N10-methylene-H4F dehydrogenase/N5,N10-methenyl-H4F cyclohydrolase) and GlyA (serine:H4F hydroxymethyltransferase) were heterologously overproduced inEscherichia coli, purified and found to be specific for methylene-H4F and H4F, respectively (apparentKm below 5 µM). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized inM. barkeri. The results thus indicate thatM. barkerishould contain H4F, which was supported by the finding that growth ofM. barkeriwas dependent on folic acid and that the vitamin could be substituted byp-aminobenzoic acid, a biosynthetic precursor of H4F. From thep-aminobenzoic acid requirement, an intracellular H4F concentration of approximately 5 µM was estimated. Evidence is presented that thep-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H4SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H4SPT and H4F inM. barkeriis in agreement with earlier isotope labeling studies indicating that there are two separate C1 pools in these methanogens. [ABSTRACT FROM AUTHOR]

The fourth reaction step of CO2-reduction to methane in methanogenic archaea is catalyzed by coenzyme F420-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd). We have structurally characterized this enzyme in the selenomethionine-labelled form from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri at 1.54 A˚ resolution using the single wavelength anomalous dispersion method for phase determination. Mtd was found to be a homohexameric protein complex that is organized as a trimer of dimers. The fold of the individual subunits is composed of two domains: a larger α,β domain and a smaller helix bundle domain with a short C-terminal β-sheet segment. In the homohexamer the α,β domains are positioned at the outside of the enzyme, whereas, the helix bundle domains assemble towards the inside to form an unusual quarternary structure with a 12-helix bundle around a 3-fold axis. No structural similarities are detectable to other enzymes with F420 and/or substituted tetrahydropterins as substrates. The substrate binding sites of F420 and methylenetetrahydromethanopterin are most likely embedded into a crevice between the domains of one subunit, their isoalloxazine and tetrahydropterin rings being placed inside a pocket formed by this crevice and a loop segment of the adjacent monomer of the dimer. Mtd revealed the highest stability at low salt concentrations of all structurally characterized enzymes from M. kandleri. This finding might be due to the compact quaternary structure that buries 36% of the monomer surface and to the large number of ion pairs. [Copyright &y& Elsevier]

Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO2 for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO2 have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C1 unit conversion bound to the cofactors tetrahydrofolate (H4F), tetrahydromethanopterin (H4MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C1 carrier, the oxidation of the cofactor-bound C1 unit and its conversion to formate, and the oxidation of formate to CO2. However, the H4MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C1 units for biosynthetic reactions (e.g., H4F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes). [ABSTRACT FROM AUTHOR]

NADP-dependent methylene-H4MPT dehydrogenase, MtdA, from Methylobacterium extorquens AM1 catalyzes the dehydrogenation of methylene-tetrahydromethanopterin and methylene-tetrahydrofolate with NADP+ as cosubstrate. The X-ray structure of MtdA with and without NADP bound was established at 1.9 A˚ resolution. The enzyme is present as a homotrimer. The α,β fold of the monomer is related to that of methylene-H4F dehydrogenases, suggesting a common evolutionary origin. The position of the active site is located within a large crevice built up by the two domains of one subunit and one domain of a second subunit. Methylene-H4MPT could be modeled into the cleft, and crucial active site residues such as Phe18, Lys256, His260, and Thr102 were identified. The molecular basis of the different substrate specificities and different catalytic demands of MtdA compared to methylene-H4F dehydrogenases are discussed. [Copyright &y& Elsevier]

Methylobacterium extorquens AM1 possesses a formyltransferase (Ftr) complex that is essential for growth in the presence of methanol and involved in formaldehyde oxidation to CO2. One of the subunits of the complex carries the catalytic site for transfer of the formyl group from tetrahydromethanopterin to methanofuran (MFR). We now found via nuclear magnetic resonance-based studies that the Ftr complex also catalyzes the hydrolysis of formyl-MFR and generates formate. The enzyme was therefore renamed Ftr/hydrolase complex (Fhc). FhcA shares a sequence pattern with amidohydrolases and is assumed to be the catalytic site where the hydrolysis takes place. [Copyright &y& Elsevier]

Methane is an end product of anaerobic degradation of organic compounds in fresh water environments such as lake sediments and the intestinal tract of animals. Methanogenic archaea produce methane from carbon dioxide and molecular hydrogen, acetate and C1 compounds such as methanol in an energy gaining process. The methanogenic pathway utilizing carbon dioxide and molecular hydrogen involves ten methanogen specific enzymes, which catalyze unique reactions using novel coenzymes. These enzymes have been purified and biochemically characterized. The genes encoding the enzymes have been cloned and sequenced. Recently, crystal structures of five methanogenic enzymes: formylmethanofuran : tetrahydromethanopterin formyltransferase, methenyltetrahydromethanopterin cyclohydrolase, methylenetetrahydromethanopterin reductase, F420H2: NADP oxidoreductase and methyl-coenzyme M reductase were reported. In this review, we describe the pathway utilizing carbon dioxide and molecular hydrogen and the catalytic mechanisms of the enzymes based on their crystal structures. [Copyright &y& Elsevier]

Recently, it has been shown that heterotrophic methylotrophic Proteobacteria contain tetrahydrofolate (H4F)- and tetrahydromethanopterin (H4MPT)-dependent enzymes. Here we report on the purification of two methylene tetrahydropterin dehydrogenases from the methylotroph Hyphomicrobium zavarzinii ZV580. Both dehydrogenases are composed of one type of subunit of 31 kDa. One of the dehydrogenases is NAD(P)-dependent and specific for methylene H4MPT (specific activity: 680 U/mg). Its N-terminal amino acid sequence showed sequence identity to NAD(P)-dependent methylene H4MPT dehydrogenase MtdB from Methylobacterium extorquens AM1. The second dehydrogenase is specific for NADP and methylene H4F (specific activity: 180 U/mg) and also exhibits methenyl H4F cyclohydrolase activity. Via N-terminal amino acid sequencing this dehydrogenase was identified as belonging to the classical bifunctional methylene H4F dehydrogenases/cyclohydrolases (FolD) found in many bacteria and eukarya. Apparently, the occurrence of methylene tetrahydrofolate and methylene tetrahydromethanopterin dehydrogenases is not uniform among different methylotrophic α-Proteobacteria. For example, FolD was not found in M. extorquens AM1, and the NADP-dependent methylene H4MPT dehydrogenase MtdA was present in the bacterium that also shows H4F activity. [ABSTRACT FROM AUTHOR]

Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylmethanofuran–tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 µm; apparent Km for H4MPT = 30 µm). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of ≈ 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a [4Fe-4S] cluster. Cytochrome c was found to be spontaneously reduced... [ABSTRACT FROM AUTHOR]

Recently it was found that the specific activity of H2-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) in Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum strain Marburg) increased six-fold when the hydrogenotrophic archaeon was grown in chemostat culture under nickel-limited conditions. We report here that the increase is due, at least in part, to increased expression of the hmd gene. This was demonstrated by Northern and Western blot analysis. These techniques were also used to show that hmd expression in growing M. marburgensis is not under the control of the H2 concentration. Studies with monoclonal antibodies on the effect of growth conditions on the expression of hmdII and hmdIII, which have been proposed to encode Hmd isoenzymes, were also carried out. The results indicate that the expression of these two genes is regulated by H2 rather than by nickel, and that HmdII and HmdIII most probably do not exhibit Hmd activity. [ABSTRACT FROM AUTHOR]

Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H4MPT) with NAD+ and NADP+. The purification of a 32-kDa NADP-specific methylene H4MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H4MPT dehydrogenase (MtdB) from this aerobic α-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H4MPT to methenyl H4MPT with NAD+ and NADP+ via a ternary complex catalytic mechanism. The Km for methylene H4MPT was 50 µm with NAD+ (Vmax = 1100 U·mg-1) and 100 µm with NADP+ (Vmax = 950 U·mg-1). The Km value for NAD+ was 200 µm and for NADP+ 20 µm. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal amino-acid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis. [ABSTRACT FROM AUTHOR]

The strictly anaerobic Archaeon Ferroglobus placidus was grown chemolithoautotrophically on H2 and nitrate and analyzed for enzymes and coenzymes possibly involved in autotrophic CO2 fixation. The following enzymes were found [values in parentheses = μmol min–1 (mg protein)–1]: formylmethanofuran dehydrogenase (0.2), formylmethanofuran:tetrahydromethanopterin formyltransferase (0.6), methenyltetrahydromethanopterin cyclohydrolase (10), F420-dependent methylenetetrahydromethanopterin dehydrogenase (1.5), F420-dependent methylenetetrahydromethanopterin reductase (0.4), and carbon monoxide dehydrogenase (0.1). The cells contained coenzyme F420 (0.4 nmol/mg protein), tetrahydromethanopterin (0.9 nmol/ mg protein), and cytochrome b (4 nmol/mg membrane protein). From the enzyme and coenzyme composition of the cells, we deduced that autotrophic CO2 fixation in F. placidus proceeds via the carbon monoxide dehydrogenase pathway as in autotrophically growing Archaeoglobus and Methanoarchaea species. Evidence is also presented that cell extracts of F. placidus catalyze the reduction of two molecules of nitrite to 1 N2O with NO as intermediate (0.1 μmol N2O formed per min and mg protein), showing that – at least in principle – F. placidus has a denitrifying capacity. [ABSTRACT FROM AUTHOR]

Archaeoglobus lithotrophicus is a hyperthermophilic Archaeon that grows on H and sulfate as energy sources and CO as sole carbon source. The autotrophic sulfate reducer was shown to contain all the enzyme activities and coenzymes of the reductive carbon monoxide dehydrogenase pathway for autotrophic CO fixation as operative in methanogenic Archaea. With the exception of carbon monoxide dehydrogenase these enzymes and coenzymes were also found in A. profundus. This organism grows lithotrophically on H and sulfate, but differs from A. lithotrophicus in that it cannot grow autotrophically: A. profundus requires acetate and CO for biosynthesis. The absence of carbon monoxide dehydrogenase in A. profundus is substantiated by the observation that this organism, in contrast to A. lithotrophicus, is not mini-methanogenic and contains only relatively low concentrations of corrinoids. [ABSTRACT FROM AUTHOR]

It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H-forming N, N-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor. We describe here the presence in this Archaeon of a second N, N-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F-dependent. This enzyme was purified and characterized. The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7±0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa. The enzyme activity increased to an optimum with increasing salt concentrations. Optimal salt concentrations were e.g. 2 M (NH)SO, 2 M NaHPO, 1.5 M KHPO, and 2 M NaCl. In the absence of salts the enzyme exhibited almost no activity. The salts affected mainly the V rather than the K of the enzyme. The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group. In the presence of M (NH)SO the V was 4000 U/mg ( k=2400 s) and the K for N, N-methylenetetrahydromethanopterin and for coenzyme F were 80 μM and 20 μM, respectively. The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM KHPO at 90°C for one hour. The N-terminal amino acid sequence was found to be similar to that of the F-dependent N, N-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus. [ABSTRACT FROM AUTHOR]

Archaeoglobus fulgidus and Methanopyrus kandleri are both extremely thermophilic Archaea with a growth temperature optimum at 83°C and 98°C, respectively. Both Archaea contain an active N, N-methenyltetrahydromethanopterin cyclohydrolase. The enzyme from M. kandleri has recently been characterized. We describe here the purification and properties of the enzyme from A. fulgidus. The cyclohydrolase from A. fulgidus was purified 180-fold to apparent homogeneity and its properties were compared with those recently published for the cyclohydrolase from M. kandleri. The two cytoplasmic enzymes were found to have very similar molecular and catalytic properties. They differed, however, significantly with respect of the effect of KHPO and of other salts on the activity and the stability. The cyclohydrolase from A. fulgidus required relatively high concentrations of KHPO (1 M) for optimal thermostability at 90°C but did not require salts for activity. Vice versa, the enzyme from M. kandleri was dependent on high KHPO concentrations (1.5 M) for optimal activity but not for thermostability. Thus the activity and structural stability of the two thermophilic enzymes depend in a completely different way on the concentration of inorganic salts. The molecular basis for these differences are discussed. [ABSTRACT FROM AUTHOR]

The sulfate-reducing Archaeoglobus fulgidus contains a number of enzymes previously thought to be unique for methanogenic Archaea. The purification and properties of two of these enzymes, of formylmethanofuran: tetrahydromethanopterin formyltransferase and of N, N-methylenetetrahydromethanopterin dehydrogenase (coenzyme F dependent) are described here. A comparison of the N-terminal amino acid sequences and of other molecular properties with those of the respective enzymes from three methanogenic Archaea revealed a high degree of similarity. [ABSTRACT FROM AUTHOR]

We measured F-dependent N,N-methylenetetrahydro-methanopterin dehydrogenase, N, N-methenyltetrahydro-methanopterin cyclohydrolase, and F-reducing hydrogenase levels in Methanosarcina barkeri grown on various substrates. Variation in dehydrogenase levels during growth on a specific substrate was usually <3-fold, and much less for cyclohydrolase. H−CO-, methanol-, and H−CO+ methanol-grown cells had roughly equivalent levels of dehydrogenase and cyclohydrolase. In acetate-grown cells cyclohydrolase level was lowered 2 to 3-fold and dehydrogenase 10 to 80-fold; this was not due to repression by acetate, since, if cultures growing on acetate were supplemented with methanol or H−CO, dehydrogenase levels increased 14 to 19-fold, and cyclohydrolase levels by 3 to 4-fold. Compared to H−CO- or methanol-grown cells, acetate-or H−CO + methanol-grown cells had lower levels of and less growth phase-dependent variation in hydrogenase activity. Our data are consistent with the following hypotheses: 1. M. barkeri oxidizes methanol via a portion of the CO-reduction pathway operated in the reverse direction. 2. When steps from CO to CH-S-CoM in the CO-reduction pathway (in either direction) are not used for methanogenesis, hydrogenase activity is lowered. [ABSTRACT FROM AUTHOR]

The activity of purified N, N-methenyltetrahydromethanopterin cyclohydrolase from Methanopyrus kandleri was found to increase up to 200-fold when potassium phosphate was added in high concentrations (1.5 M) to the assay. A 200-fold stimulation was also observed with sodium phosphate (1 M) and sodium sulfate (1 M) whereas stimulation by potassium sulfate (0.8 M), ammonium sulfate (1.5 M), potassium chloride (2.5 M), and sodium chloride (2 M) was maximal 100-fold. A detailed kinetic analysis of the effect of potassium phosphate revealed that this salt exerted its stimulatory effect by decreasing the K for N, N-methenyltetrahydromethanopterin from 2 mM to 40 μM and by increasing the V from 2000 U/mg (k=1385 s) to 13300 U/mg (k=9200 s). Besides increasing the catalytic efficiency (k/ K) salts were found to protect the cyclohydrolase from heat inactivation. For maximal thermostability much lower concentrations (0.1 M) of salts were required than for maximal activity. [ABSTRACT FROM AUTHOR]

Methylene-HMPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a K for methylene-HMPT of 16 μM, a K for FH of 4 μM, and a V of 450 U/mg (K=265 s) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F (0.2 mM), methylene-HMPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-HMPT reductase (F-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described. [ABSTRACT FROM AUTHOR]

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H and CO and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two α-, β- and γ-subunits and that it contained the nickel porphinoid coenzyme F as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the γ-subunit was determined. A comparison with the N-terminal sequences of the γ-subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity. Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO (apparent V at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N,N-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N,N-methylenetetrahydromethanopterin dehydrogenase (H-forming), 33 U/mg; N,N-methylenetetrahydromethanopterin reductase (coenzyme F dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent K values for these enzymes and the effect of salts on their activities were determined. The coenzyme F present in M. kandleri was identified as coenzyme F-2 with 2 γ-glutamyl residues. [ABSTRACT FROM AUTHOR]

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H and CO and that shows no close phylogenetic relationship to any methanogens known so far. N N-Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogenesis from CO, was purified from this hyperthermophile. The apparent molecular mass of the native enzyme was found to be 300 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only one polypeptide of apparent molecular mass 38 kDa. The ultraviolet/visible spectrum of the enzyme was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was specific for reduced coenzyme F as electron donor; NADH, NADPH or reduced dyes could not substitute for the 5-deazaflavin. The catalytic mechanism was found to be of the ternary complex type as deduced from initial velocity plots. V at 65°C and pH 6.8 was 435 U/mg (k=275 s) and the K for methylenetetrahydro-methanopterin and for reduced F were 6 μM and 4 μM, respectively. From Arrhenius plots an activation energy of 34 kJ/mol was determined. The Q between 40°C and 90°C was 1.5. The reductase activity was found to be stimulated over 100-fold by sulfate and by phosphate. Maximal stimulation (100-fold) was observed at a sulfate concentration of 2.2 M and at a phosphate concentration of 2.5 M. Sodium-, potassium-, and ammonium salts of these anions were equally effective. Chloride, however, could not substitute for sulfate or phosphate in stimulating the enzyme activity. The thermostability of the reductase was found to be very low in the absence of salts. In their presence, however, the reductase was highly thermostable. Salt concentrations between 0.1 M and 1.5 M were required for maximal stability. Potassium salts proved more effective than ammonium salts, and the latter more effective than sodium salts in stabilizing the enzyme activity. The anion was of less importance. The N-terminal amino acid sequence of the reductase from M. kandleri was determined and compared with that of the enzyme from Methanobacterium thermoautotrophicum and Methanosarcina barkeri. Significant similarity was found. [ABSTRACT FROM AUTHOR]

The activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase were tested in cell extracts of 10 different methanogenic bacteria grown on H/CO or on other methanogenic substrates. The four activities were found in all the organisms investigated: Methanobacterium thermoautotrophicum,M. wolfei, Methanobrevibacter arboriphilus, Methanosphaera stadtmanae, Methanosarcina barkeri (strains Fusaro and MS), Methanothrix soehngenii, Methanospirillum hungatei, Methanogenium organophilum, and Methanococcus voltae. Cell extracts of H/CO grown M. barkeri and of methanol grown M. barkeri showed the same specific activities suggesting that the four enzymes are of equal importance in CO reduction to methane and in methanol disproportionation to CO and CH. In contrast, cell extracts of acetate grown M. barkeri exhibited much lower activities of formylmethanofuran dehydrogenase and methylenetetrahydromethanopterin dehydrogenase suggesting that these two enzymes are not involved in methanogenesis from acetate. In M. stadtmanae, which grows on H and methanol, only heterodisulfide reductase was detected in activities sufficient to account for the in vivo methane formation rate. This finding is consistent with the view that the three other oxidoreductases are not required for methanol reduction to methane with H. [ABSTRACT FROM AUTHOR]

The dehydrogenation of N , N -methylenetetrahydromethanopterin (CH=HMPT) to N , N -methenyltetrahydromethanopterin (CH≡HMPT) is an intermediate step in the oxidation of methanol to CO in Methanosarcina barkeri. The reaction is catalyzed by CH=HMPT dehydrogenase, which was found to be specific for coenzyme F as electron acceptor; neither NAD, NADP nor viologen dyes could substitute for the 5-deazaflavin. The dehydrogenase was anaerobically purified almost 90-fold to apparent homogeneity in a 32% yield by anion exchange chromatography on DEAE Sepharose and Mono Q HR, and by affinity chromatography on Blue Sepharose. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band with an apparent mass of 31 kDa. The apparent molecular mass of the native enzyme determined by polyacrylamide gradient gel electrophoresis was 240 kDa. The ultraviolet/visible spectrum of the purified enzyme was almost identical to that of albumin suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentrations were linear: the apparent K for CH=HMPT and for coenzyme F were found to be 6 μM and 25 μM, respectively. V was 4,000 μmol min·mg protein (k=2,066 s) at pH 6 (the pH optimum) and 37°C. The Arrhenius activation energy was 40 kJ/mol. The N-terminal amino acid sequence was found to be 50% identical with that of the F-dependent CH=HMPT dehydrogenase isolated from H/CO grown Methanobacterium thermoautotrophicum. [ABSTRACT FROM AUTHOR]

Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO and HS. The organism contains coenzyme F, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C-intermediates otherwise only used by methanogenic bacteria. [ABSTRACT FROM AUTHOR]

Serine transhydroxymethylase of Methanobacterium thermoautotrophicum has been purified to within 95% of homogeneity. Activity was strictly dependent on tetrahydromethanopterin, tetrahydrofolate being unable to serve as the acceptor C units from l-serine. The native protein has a molecular weight of about 102,000 daltons. The enzyme shows maximal activity at 60°C, has a pH optimum of 8.1, and required pyridoxal-5′-phosphate and Mg for optimal activity. [ABSTRACT FROM AUTHOR]