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Asgard is an archaeal superphylum that might hold the key to understand the origin of eukaryotes, but its diversity and ecological roles remain poorly understood. Here, we reconstructed 15 metagenomic-assembled genomes from coastal sediments covering most known Asgard archaea and a novel group, which is proposed as a new Asgard phylum named as the “Gerdarchaeota”. Genomic analyses predict that Gerdarchaeota are facultative anaerobes in utilizing both organic and inorganic carbon. Unlike their closest relatives Heimdallarchaeota, Gerdarchaeota have genes encoding for cellulase and enzymes involved in the tetrahydromethanopterin-based Wood–Ljungdahl pathway. Transcriptomics showed that most of our identified Asgard archaea are capable of degrading organic matter, including peptides, amino acids and fatty acids, occupying ecological niches in different depths of layers of the sediments. Overall, this study broadens the diversity of the mysterious Asgard archaea and provides evidence for their ecological roles in coastal sediments.

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth’s diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H2), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C1-carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F420H2 re-oxidation) as found for methylotrophic methanogenesis.

The reutilisation of food waste for the production of clean energy was promoted by supplementing magnet powder in anaerobic digestion (AD). This study found that adding 5% magnet powder optimally increased the amount of biogas produced by 61.9%, and the pH and volatile fatty acids (VFA) content had the greatest correlation with biogas production. A further metagenomics analysis in the early, middle, and late stages of the AD revealed that interaction between bacteria and archaea had highest explanation rate for pH and VFA changes rather than enzymes. Moreover, the 5% magnet powder increased the proportion of the CO2 methanogenesis and decreased the acetate methanogenesis on day 15 of peak biogas production. And it was an innovative discovery that conversion of tetrahydromethanopterin S-methyltransferase to methane increased, which is an important common node of methanogenesis metabolic and may be the fundamental reason for the increase in biogas production caused by magnetic powder.

Symbiotic digestion of lignocellulose in the hindgut of higher termites is mediated by a diverse assemblage of bacteria and archaea. During a large-scale metagenomic study, we reconstructed 15 metagenome-assembled genomes of Bathyarchaeia that represent two distinct lineages in subgroup 6 (formerly MCG-6) unique to termite guts. One lineage (TB2; Candidatus Termitimicrobium) encodes all enzymes required for reductive acetogenesis from CO2 via an archaeal variant of the Wood–Ljungdahl pathway, involving tetrahydromethanopterin as C1 carrier and an (ADP-forming) acetyl-CoA synthase. This includes a novel 11-subunit hydrogenase, which possesses the genomic architecture of the respiratory Fpo-complex of other archaea but whose catalytic subunit is phylogenetically related to and shares the conserved [NiFe] cofactor-binding motif with [NiFe] hydrogenases of subgroup 4 g. We propose that this novel Fpo-like hydrogenase provides part of the reduced ferredoxin required for CO2 reduction and is driven by the electrochemical membrane potential generated from the ATP conserved by substrate-level phosphorylation; the other part may require the oxidation of organic electron donors, which would make members of TB2 mixotrophic acetogens. Members of the other lineage (TB1; Candidatus Termiticorpusculum) are definitely organotrophic because they consistently lack hydrogenases and/or methylene-tetrahydromethanopterin reductase, a key enzyme of the archaeal Wood–Ljungdahl pathway. Both lineages have the genomic capacity to reduce ferredoxin by oxidizing amino acids and might conduct methylotrophic acetogenesis using unidentified methylated compound(s). Our results indicate that Bathyarchaeia of subgroup 6 contribute to acetate formation in the guts of higher termites and substantiate the genomic evidence for reductive acetogenesis from organic substrates, possibly including methylated compounds, in other uncultured representatives of the phylum.

Methanogens are considered as one of the earliest life forms on Earth, and together with anaerobic methane-oxidizing archaea, they have crucial effects on climate stability. Yet, the origin and evolution of anaerobic alkane metabolism in the domain Archaea remain controversial. Here, we show that methanogenesis was already present in the common ancestor of Euryarchaeota, TACK archaea, and Asgard archaea likely in the late Hadean or early Archean eon and that the ancestral methanogen was dependent on methylated compounds and hydrogen. Carbon dioxide-reducing methanogenesis developed later through the evolution of tetrahydromethanopterin S-methyltransferase, which linked methanogenesis to the Wood-Ljungdahl pathway for energy conservation. Multicarbon alkane metabolisms in Archaea also originated early, with genes coding for the activation of short- or even long-chain alkanes likely evolving from an ethane-metabolizing ancestor. These genes were likely horizontally transferred to multiple archaeal clades including Candidatus (Ca) Bathyarchaeota, Ca. Helarchaeota, Ca Hadesarchaeota, and the methanogenic Ca. Methanoliparia.

The sulfate-dependent, anaerobic oxidation of methane (AOM) is an important sink for methane in marine environments. It is carried out between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) living in syntrophic partnership. In this study, we compared the genomes, gene expression patterns and ultrastructures of three phylogenetically different microbial consortia found in hydrocarbon-rich environments under different temperature regimes: ANME-1a/HotSeep-1 (60°C), ANME-1a/Seep-SRB2 (37°C) and ANME-2c/Seep-SRB2 (20°C). All three ANME encode a reverse methanogenesis pathway: ANME-2c encodes all enzymes, while ANME-1a lacks the gene for N5,N10-methylene tetrahydromethanopterin reductase (mer) and encodes a methylenetetrahydrofolate reductase (Met). The bacterial partners contain the genes encoding the canonical dissimilatory sulfate reduction pathway. During AOM, all three consortia types highly expressed genes encoding for the formation of flagella or type IV pili and/or c-type cytochromes, some predicted to be extracellular. ANME-2c expressed potentially extracellular cytochromes with up to 32 hemes, whereas ANME-1a and SRB expressed less complex cytochromes (≤ 8 and ≤ 12 heme respectively). The intercellular space of all consortia showed nanowire-like structures and heme-rich areas. These features are proposed to enable interspecies electron exchange, hence suggesting that direct electron transfer is a common mechanism to sulfate-dependent AOM, and that both partners synthesize molecules to enable it.

Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-H4MPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotroph Methylorubrum extorquens and identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations., Proceedings of the National Academy of Sciences of the United States of America, 116 (51), ISSN:0027-8424, ISSN:1091-6490

Dimethylsulfide is a volatile organic sulfur compound that provides the largest input of biogenic sulfur from the oceans to the atmosphere, and thence back to land, constituting an important link in the global sulfur cycle. Microorganisms degrading DMS affect fluxes of DMS in the environment, but the underlying metabolic pathways are still poorly understood. Methylophaga thiooxydans is a marine methylotrophic bacterium capable of growth on DMS as sole source of carbon and energy. Using proteomics and transcriptomics we identified genes expressed during growth on dimethylsulfide and methanol to refine our knowledge of the metabolic pathways that are involved in DMS and methanol degradation in this strain. Amongst the most highly expressed genes on DMS were the two methanethiol oxidases driving the oxidation of this reactive and toxic intermediate of DMS metabolism. Growth on DMS also increased expression of the enzymes of the tetrahydrofolate linked pathway of formaldehyde oxidation, in addition to the tetrahydromethanopterin linked pathway. Key enzymes of the inorganic sulfur oxidation pathway included flavocytochrome c sulfide dehydrogenase, sulfide quinone oxidoreductase, and persulfide dioxygenases. A sulP permease was also expressed during growth on DMS. Proteomics and transcriptomics also identified a number of highly expressed proteins and gene products whose function is currently not understood. As the identity of some enzymes of organic and inorganic sulfur metabolism previously detected in Methylophaga has not been characterized at the genetic level yet, highly expressed uncharacterized genes provide new targets for further biochemical and genetic analysis. A pan-genome analysis of six available Methylophaga genomes showed that only two of the six investigated strains, M. thiooxydans and M. sulfidovorans have the gene encoding methanethiol oxidase, suggesting that growth on methylated sulfur compounds of M. aminisulfidivorans is likely to involve different enzymes and metabolic intermediates. Hence, the pathways of DMS-utilization and subsequent C1 and sulfur oxidation are not conserved across Methylophaga isolates that degrade methylated sulfur compounds.

The effect of different bioaugmentation strategies on anaerobic digestion related to the alleviation of ammonia inhibition was investigated in a long-term operation. The long-term operation confirmed that bioaugmentation is a stable method. A 35% increase in methane production (MP) was observed in bottles bioaugmented with Methanosarcina barkeri (MSB) or Syntrophaceticu schinkii (SS) + Methanobrevibacter smithii (MBS), and a 49% increment was obtained from the bottles bioaugmented with Methanosaeta harundinacea (MSH) + SS + MBS. Results suggest that the enhancement in both aceticlastic and hydrogenotrophic methanogenic pathways should be considered, and bioaugmentation strain should be properly selected to achieve a synergistic effect. The microbial community analysis indicated Methanosarcina spp. was the dominant archaea. Combined with specific methanogenic activity and carbon isotope fractionation analysis, it was suggested that Methanosarcina spp. performed differently in methanogenic pathways in different bottles. The abundance of COG and total enzymes in the bottles with high MP (MSB and MSH + SS + MBS) was higher than that in the other bottles. The ratio of the functional enzyme tetrahydromethanopterin S-methyltransferase subunit H (EC 2.1.1.86) to formylmethanofuran dehydrogenase subunit E (EC 1.2.99.5) and the relative abundance of enolase (EC 1.2.1.2) confirmed the aceticlastic methanogenic pathway of MSB in Group 1 and the pathway enhancement balance in MSH + SS + MBS. The modified Anaerobic Digestion Model No.1 including syntrophic acetate oxidation was used for simulation with R2 > 0.96. Simulated contribution rate data indicated that the hydrogenotrophic methanogenic pathway was dominant while both two pathways got strengthened, which is consistent with the findings of the microbial analysis.

Lanthanides (Ln) are an essential cofactor for XoxF-type methanol dehydrogenases (MDHs) in Gram-negative methylotrophs. The Ln3+ dependency of XoxF has expanded knowledge and raised new questions in methylotrophy, including the differences in characteristics of XoxF-type MDHs, their regulation, and the methylotrophic metabolism including formaldehyde oxidation. In this study, we genetically identified one set of Ln3+- and Ca2+-dependent MDHs (XoxF1 and MxaFI), that are involved in methylotrophy, and an ExaF-type Ln3+-dependent ethanol dehydrogenase, among six MDH-like genes in Methylobacterium aquaticum strain 22A. We also identified the causative mutations in MxbD, a sensor kinase necessary for mxaF expression and xoxF1 repression, for suppressive phenotypes in xoxF1 mutants defective in methanol growth even in the absence of Ln3+. Furthermore, we examined the phenotypes of a series of formaldehyde oxidation-pathway mutants (fae1, fae2, mch in the tetrahydromethanopterin (H4MPT) pathway and hgd in the glutathione-dependent formaldehyde dehydrogenase (GSH) pathway). We found that MxaF produces formaldehyde to a toxic level in the absence of the formaldehyde oxidation pathways and that either XoxF1 or ExaF can oxidize formaldehyde to alleviate formaldehyde toxicity in vivo. Furthermore, the GSH pathway has a supportive role for the net formaldehyde oxidation in addition to the H4MPT pathway that has primary importance. Studies on methylotrophy in Methylobacterium species have a long history, and this study provides further insights into genetic and physiological diversity and the differences in methylotrophy within the plant-colonizing methylotrophs.

Methane-producing archaea and methylotrophic bacteria use tetrahydromethanopterin (H4MPT) and/or tetrahydrofolate (H4F) as coenzymes in one-carbon (C1) transfer pathways. The α-proteobacterium Methylobacterium extorquens AM1 contains a dihydromethanopterin reductase (DmrA) and two annotated dihydrofolate reductases (DfrA and DfrB). DmrA has been shown to catalyze the final step of H4MPT biosynthesis; however, the functions of DfrA and DfrB have not been examined biochemically. Moreover, sequence alignment (BLAST) searches have recognized scores of proteins that share up to 99% identity with DmrA but are annotated as diacylglycerol kinases (DAGK). In this work, we used bioinformatics and enzyme assays to provide insight into the phylogeny and substrate specificity of selected Dfr and DmrA homologs. In a phylogenetic tree, DmrA and homologs annotated as DAGKs grouped together in one clade. Purified histidine-tagged versions of the annotated DAGKs from Hyphomicrobium nitrativorans and Methylobacterium nodulans (respectively, sharing 69% and 84% identity with DmrA) showed only low activity in phosphorylating 1,2-dihexanoyl-sn-glycerol when compared with a commercial DAGK from E. coli. However, the annotated DAGKs successfully reduced a dihydromethanopterin analog (dihydrosarcinapterin, H2SPT) with kinetic values similar to those determined for M. extorquens AM1 DmrA. DfrA and DfrB showed little or no ability to reduce H2SPT under the conditions studied; however, both catalyzed the NADPH-dependent reduction of dihydrofolate. These results provide the first evidence that DfrA and DfrB function as authentic dihydrofolate reductases, while DAGKs with greater than 69% identity to DmrA may be misannotated and are likely to function in H4MPT biosynthesis.

Asgard is a newly proposed archaeal superphylum. Phylogenetic position of Asgard archaea and its relationships to the origin of eukaryotes is attracting increasingly research interest. However, in-depth knowledge of their diversity, distribution, and activity of Asgard archaea remains limited. Here, we used phylogenetic analysis to cluster the publicly available Asgard archaeal 16S rRNA gene sequences into 13 subgroups, including five previously unknown subgroups. These lineages were widely distributed in anaerobic environments, with the majority of 16S rRNA gene sequences (92%) originating from sediment habitats. Co-occurrence analysis revealed potential relationships between Asgard, Bathyarchaeota, and Marine Benthic Group D archaea. Genomic analysis suggested that Asgard archaea are potentially mixotrophic microbes with divergent metabolic capabilities. Importantly, metatranscriptomics confirmed the versatile lifestyles of Lokiarchaeota and Thorarchaeota, which can fix CO2using the tetrahydromethanopterin Wood-Ljungdahl pathway, perform acetogenesis, and degrade organic matters. Overall, this study broadens the understandings of Asgard archaea ecology, and also provides the first evidence to support a transcriptionally active mixotrophic lifestyle of Asgard archaea, shedding light on the potential roles of these microorganisms in the global biogeochemical cycling.

The metabolic pathways of one-carbon compounds utilized by colorless sulfur bacterium Beggiatoa leptomitoformis D-402 were revealed based on comprehensive analysis of its genomic organization, together with physiological, biochemical and molecular biological approaches. Strain D-402 was capable of aerobic methylotrophic growth with methanol as a sole source of carbon and energy and was not capable of methanotrophic growth because of the absence of genes of methane monooxygenases. It was established that methanol can be oxidized to CO(2) in three consecutive stages. On the first stage methanol was oxidized to formaldehyde by the two PQQ (pyrroloquinolinequinone)-dependent methanol dehydrogenases (MDH): XoxF and Mdh2. Formaldehyde was further oxidized to formate via the tetrahydromethanopterin (H(4)MPT) pathway. And on the third stage formate was converted to CO(2) by NAD(+)-dependent formate dehydrogenase Fdh2. Finally, it was established that endogenous CO(2), formed as a result of methanol oxidation, was subsequently assimilated for anabolism through the Calvin–Benson–Bassham cycle. The similar way of one-carbon compounds utilization also exists in representatives of another freshwater Beggiatoa species—B. alba. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12088-018-0737-x) contains supplementary material, which is available to authorized users.

We sequenced the genomes of 19 methylotrophic isolates from Lake Washington, which belong to nine genera within eight families of the Alphaproteobacteria, two of the families being the newly proposed families. Comparative genomic analysis with a focus on methylotrophy metabolism classifies these strains into heterotrophic and obligately or facultatively autotrophic methylotrophs. The most persistent metabolic modules enabling methylotrophy within this group are the N-methylglutamate pathway, the two types of methanol dehydrogenase (MxaFI and XoxF), the tetrahydromethanopterin pathway for formaldehyde oxidation, the serine cycle and the ethylmalonyl-CoA pathway. At the same time, a great potential for metabolic flexibility within this group is uncovered, with different combinations of these modules present. Phylogenetic analysis of key methylotrophy functions reveals that the serine cycle must have evolved independently in at least four lineages of Alphaproteobacteria and that all methylotrophy modules seem to be prone to lateral transfers as well as deletions.

Prior studies have indicated that MJ1099 fromMethanocaldococcus jannaschiihas roles in the biosynthesis of tetrahydromethanopterin and methanofuran, two key cofactors of one-carbon (C1) metabolism in diverse organisms including the methanogenic archaea. Here, the structure of MJ1099 has been solved to 1.7 Å resolution using anomalous scattering methods. The results indicate that MJ1099 is a member of the TIM-barrel superfamily and that it is a homohexamer. Bioinformatic analyses identified a potential active site that is highly conserved among MJ1099 homologs and the key amino acids involved were identified. The results presented here should guide further studies of MJ1099 including mechanistic studies and possibly the development of inhibitors that target the methanogenic archaea in the digestive tracts of humans and that are a source of the greenhouse gas methane.

Metabolic Origins Methyl groups, including those derived from or ending up in methane, are managed through a dizzying array of metabolic pathways. Some of these originated early in life's history, and their evolution paralleled and contributed to Earth's chemistry. Adam et al. analyzed a set of bacterial and archaeal genomes, looking for patterns in the presence and divergence of enzymes in the tetrahydromethanopterin methyl branch of the Wood–Ljungdahl pathway, which is involved in carbon redox reactions. Expanding previous analyses narrowed the distance between bacterial and archaeal sequences, making horizontal transfer more plausible relative to an origin in the last universal common ancestor. An origin of this branch in Archaea is likely given wide distribution therein and enzyme phylogenies consistent with those of the organisms. Nat. Microbiol. 10.1038/s41564-019-0534-2 (2019).

In this opinion article, I wish to highlight the fact that reactions linked to tetrahydromethanopterin (H4MPT) and methanofuran (MF), the ones involved in methanogenesis as well as in methylotrophy, are much more widespread among both Bacteria and Archaea than originally thought. While, over the past two decades, databases of the respective genes have been steadily growing and expanding to include novel, divergent sequences, belonging to a variety of taxa, somehow a view still prevails of the limited distribution of these genes, along with an evolutionary scenario in which genes for the methanogenesis pathway were horizontally transferred from Euryarchaea into Proteobacteria (Graham et al., 2000; Gogarten et al., 2002; Boucher et al., 2003; Braakman and Smith, 2012; Arnold, 2015). The two main arguments originally used to support this scenario were (1) the limited distribution of the H4MPT/MF-dependent pathway in the bacterial domain of life, and (2) the low probability of the respective genes being lost in most bacterial lineages (Boucher et al., 2003). However, these arguments can be easily refuted in the light of the current knowledge. In Figure ​Figure1,1, I utilize the recently constructed universal tree of life (Hug et al., 2016), to map the taxa in which at least some of the genes for the H4MPT/MF-dependent C1 transfers are recognized. Among the Archaea, these include, in addition to the well-characterized methanonogens or methane oxidizers, members of Euryarchaeota not known for a methanogenic life style (Thermoplasmatales, Hadesarchaea; Baker et al., 2016), members of Crenarchaeota (Thermoproteales, Ignisphaera, Ingnispaeroid; Goker et al., 2010; Jay et al., 2016), Bathyarchaeota (Evans et al., 2015; Lazar et al., 2016), and Thorarchaeota (Seitz et al., 2016). Among the Bacteria, genes for the H4MPT/MF-dependent reactions have been identified, beside Alpha-, Beta-, and Gammaproteobacteria (Vorholt et al., 1999), in the genomes of Planctomycetes (Chistoserdova et al., 2004; Chistoserdova, 2013), Deltaproteobacteria, Firmicutes, Actinomycetes, Synergistetes, Chloroflexi (Brown et al., 2011 and unpublished genomes available through the NCBI), as well as in the Candidate phylum NC10 (Ettwig et al., 2010). This wide distribution across the tree of life (Figure ​(Figure1),1), along with great sequence divergence for the genes in question (Chistoserdova, 2013; Evans et al., 2015; Spang et al., 2015) support a scenario of a long evolution within both Archaea and Bacteria, and point to the emergence of these reactions in early life, before Bacteria and Archaea have branched apart.

Methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) cyclohydrolase (Mch) catalyzes the interconversion of methenyl-H(4)MPT(+) and formyl-H(4)MPT in the one-carbon energy metabolism of methanogenic, methanotrophic, and sulfate-reducing archaea and of methylotrophic bacteria. To understand the catalytic mechanism of this reaction, we kinetically characterized site-specific variants of Mch from Archaeoglobus fulgidus (aMch) and determined the X-ray structures of the substrate-free aMch(E186Q), the aMch:H(4)MPT complex, and the aMch(E186Q):formyl-H(4)MPT complex. (Formyl-)H(4)MPT is embedded inside a largely preformed, interdomain pocket of the homotrimeric enzyme with the pterin and benzyl rings being oriented nearly perpendicular to each other. The active site is primarily built up by the segment 93:95, Arg183 and Glu186 that either interact with the catalytic water attacking methenyl-H(4)MPT(+) or with the formyl oxygen of formyl-H(4)MPT. The catalytic function of the strictly conserved Arg183 and Glu186 was substantiated by the low enzymatic activities of the E186A, E186D, E186N, E186Q, R183A, R183Q, R183E, R183K, and R183E-E186Q variants. Glu186 most likely acts as a general base. Arg183 decisively influences the pK(a) value of Glu186 and the proposed catalytic water mainly by its positive charge. In addition, Glu186 appears to be also responsible for product specificity by donating a proton to the directly neighbored N(10) tertiary amine of H(4)MPT. Thus, N(10) becomes a better leaving group than N(5) which implies the generation of N(5)-formyl-H(4)MPT. For comparison, methenyltetrahydrofolate (H(4)F) cyclohydrolase produces N(10)-formyl-H(4)F in an analogous reaction. An enzymatic mechanism of Mch is postulated and compared with that of other cyclohydrolases.

Methanopyrus kandleri is a methanogenic Archaeon that grows on H2 and CO2 at a temperature optimum of 98 degrees C. The gene ftr encoding the formylmethanofuran:tetrahydromethanopterin formyltransferase, an enzyme involved in CO2 reduction to methane, has been cloned, sequenced, and overexpressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% by simply heating the cell extract to 90 degrees C in 1.5 M K2HPO4 pH 8.0 for 30 min. From 1 g wet cells (70 mg protein) approximately 14 mg formyltransferase was obtained. The purified enzyme showed essentially the same catalytic properties as that purified from M. kandleri cells. The primary structure and properties of the formyltransferase are compared with those of the enzyme from Methanobacterium thermoautotrophicum (growth temperature optimum 65 degrees C) and Methanothermus fervidus (83 degrees C). Of the three enzymes that from M. kandleri had the lowest isoelectric point (4.2) and the lowest hydrophobicity of the amino acid composition. The enzyme from M. kandleri had the relatively highest content in alanine, glutamate and glutamine and the relatively lowest content in isoleucine, leucine and lysine. These properties, some of which are unusual for enzymes from other hyperthermophilic organisms, may reflect that the formyltransferase from M. kandleri is adapted to both hyperthermophilic and halophilic conditions.

The non-methane-utilizing methylotroph, Methylophilus sp. TWE2, was isolated from tap-water during the enrichment of methanotrophs with methane. The complete genome sequence of strain TWE2 showed that this bacterium may convert methanol to formaldehyde via catalysis of methanol dehydrogenase (MDH), after which formaldehyde would be assimilated to biomass through the ribulose monophosphate (RuMP) pathway or dissimilated via the tetrahydromethanopterin (H4MPT) pathway. The deficiency of glycolysis and the TCA cycle indicate that strain TWE2 may be an obligate methylotroph. This is the first complete genome sequence of the genus Methylophilus.

Tetrahydromethanopterin (H4 MPT) is a tetrahydrofolate analogue involved as a C1 carrier in the metabolism of various groups of microorganisms. How H4MPT is bound to the respective C1 unit converting enzymes remained elusive. We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 angstrom without and at 1.9 angstrom with methylene-H4MPT bound. Methylene-H4MPT is bound in an "S"-shaped conformation into the cleft formed between two adjacent subunits. Coenzyme binding is accompanied by side chain rearrangements up to 5 angstrom and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster, and an inversion of the amide side chain of Gln88. Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70 degrees that is more pronounced than that reported for free methylene-H4MPT in solution (50 degrees). Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyzes the formation of methylene-H4MPT from H4MPT and formaldehyde. The molecular mechanism ofthis reaction involving His22 as acid catalyst is discussed.

During growth on one-carbon (C 1 ) compounds, the aerobic α-proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H 4 MPT) derivative dephospho-H 4 MPT as a C 1 carrier in addition to tetrahydrofolate. The enzymes involved in dephospho-H 4 MPT biosynthesis have not been identified in bacteria. In archaea, the final step in the proposed pathway of H 4 MPT biosynthesis is the reduction of dihydromethanopterin (H 2 MPT) to H 4 MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase. A gene encoding a dihydrofolate reductase homolog has previously been reported for M. extorquens and assigned as the putative H 2 MPT reductase gene ( dmrA ). In the present work, we describe the biochemical characterization of H 2 MPT reductase (DmrA), which is encoded by dmrA . The gene was expressed with a six-histidine tag in Escherichia coli , and the recombinant protein was purified by nickel affinity chromatography and gel filtration. Purified DmrA catalyzed the NAD(P)H-dependent reduction of H 2 MPT with a specific activity of 2.8 μmol of NADPH oxidized per min per mg of protein at 30°C and pH 5.3. Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8. While the existence of an H 2 MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea. Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase. This may be a consequence of different electron donors, NAD(P)H versus reduced F 420 , used, respectively, in bacteria and methanogenic archaea.

An enzymatic assay was developed to measure tetrahydromethanopterin (H 4 MPT) levels in wild-type and mutant cells of Methylobacterium extorquens AM1. H 4 MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H 4 MPT biosynthesis. The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene 5′-phosphate synthase, the first committed step of H 4 MPT biosynthesis. These results provide the first biochemical evidence for H 4 MPT biosynthesis genes in bacteria.

In methylotrophic bacteria, formaldehyde is an important but potentially toxic metabolic intermediate that can be assimilated into biomass or oxidized to yield energy. Previously reported was the purification of an NAD(P)(+)-dependent formaldehyde dehydrogenase (FDH) from the obligate methane-oxidizing methylotroph Methylococcus capsulatus (Bath), presumably important in formaldehyde oxidation, which required a heat-stable factor (known as the modifin) for FDH activity. Here, the major protein component of this FDH preparation was shown by biophysical techniques to comprise subunits of 64 and 8 kDa in an alpha(2)beta(2) arrangement. N-terminal sequencing of the subunits of FDH, together with enzymological characterization, showed that the alpha(2)beta(2) tetramer was a quinoprotein methanol dehydrogenase of the type found in other methylotrophs. The FDH preparations were shown to contain a highly active NAD(P)(+)-dependent methylene tetrahydromethanopterin dehydrogenase that was the probable source of the NAD(P)(+)-dependent formaldehyde oxidation activity. These results support previous findings that methylotrophs possess multiple pathways for formaldehyde dissimilation.

The classic scheme of energy metabolism during methylotrophic growth involves a formate oxidation step except in strains in which all formaldehyde is oxidized in the cyclic ribulose monophosphate cycle (1). Formate dehydrogenase (FDH) activity has been detected in most methylotrophs (3, 10, 13, 15, 22, 42), and a few FDHs have been purified and analyzed (reviewed in reference 39). In the methylotrophic yeast Candida boidinii, the FDH step was shown not to be essential for methylotrophic growth, but FDH mutants showed reduced growth on methanol (32). However, as the complete C. boidinii genome sequence is not available, the presence of other FDHs is not excluded. Mutant-based analysis of the role of the FDH step in C1 oxidation has not yet been attempted in methylotrophic bacteria. M. extorquens AM1 offers a convenient model to study this question. It possesses two pathways in which formaldehyde can be oxidized to formate (Fig. ​(Fig.1),1), one linked to tetrahydromethanopterin (H4MPT) and another linked to tetrahydrofolate (H4F) (5, 6). The enzymes involved in the two pathways have been studied in detail, and current evidence suggests that the main pathway for oxidizing formaldehyde is the H4MPT-linked pathway (reviewed in reference 37). It has been demonstrated recently that this pathway produces formate as an intermediate, a result of a formylmethanofuran transferase/hydrolase reaction (29), and thus in this pathway one molecule of formate is formed in M. extorquens AM1 per oxidized molecule of a C1 substrate, such as methanol or methylamine. This formate is subsequently oxidized to CO2, presumably by FDH (Fig. ​(Fig.11). FIG. 1. C1 metabolism of M. extorquens AM1. H4MPT, tetrahydromethanopterin; H4F, tetrahydrofolate; Fae, H4MPT-dependent formaldehyde activating enzyme (39); MtdA, NADP-dependent methylene-H4MPT dehydrogenase (8, 38); MtdB, NAD(P)-dependent methylene-H4MPT dehydrogenase ... An FDH from M. extorquens AM1 has recently been purified and characterized and shown to be a novel, tungsten-containing FDH encoded by two genes, fdh1AB (20). In this study we identified two new regions in the M. extorquens AM1 chromosome coding for two additional FDH enzymes. Using mutation analysis, we demonstrate that all three enzymes are expressed during growth on C1 compounds but none is essential for growth on methanol, providing new insight into the energetics of C1 metabolism in serine cycle methylotrophs.

The serine cycle methylotroph Methylobacterium extorquens AM1 contains two pterin-dependent pathways for C 1 transfers, the tetrahydrofolate (H 4 F) pathway and the tetrahydromethanopterin (H 4 MPT) pathway, and both are required for growth on C 1 compounds. With the exception of formate-tetrahydrofolate ligase (FtfL, alternatively termed formyl-H 4 F synthetase), all of the genes encoding the enzymes comprising these two pathways have been identified, and the corresponding gene products have been purified and characterized. We present here the purification and characterization of FtfL from M. extorquens AM1 and the confirmation that this enzyme is encoded by an ftfL homolog identified previously through transposon mutagenesis. Phenotypic analyses of the ftfL mutant strain demonstrated that FtfL activity is required for growth on C 1 compounds. Unlike mutants defective for the H 4 MPT pathway, the ftfL mutant strain does not exhibit phenotypes indicative of defective formaldehyde oxidation. Furthermore, the ftfL mutant strain remained competent for wild-type conversion of [ 14 C]methanol to [ 14 C]CO 2 . Collectively, these data confirm our previous presumptions that the H 4 F pathway is not the key formaldehyde oxidation pathway in M. extorquens AM1. Rather, our data suggest an alternative model for the role of the H 4 F pathway in this organism in which it functions to convert formate to methylene H 4 F for assimilatory metabolism.

The facultative methylotroph Methylobacterium extorquens AM1 possesses two pterin-dependent pathways for C 1 transfer between formaldehyde and formate, the tetrahydrofolate (H 4 F)-linked pathway and the tetrahydromethanopterin (H 4 MPT)-linked pathway. Both pathways are required for growth on C 1 substrates; however, mutants defective for the H 4 MPT pathway reveal a unique phenotype of being inhibited by methanol during growth on multicarbon compounds such as succinate. It has been previously proposed that this methanol-sensitive phenotype is due to the inability to effectively detoxify formaldehyde produced from methanol. Here we present a comparative physiological characterization of four mutants defective in the H 4 MPT pathway and place them into three different phenotypic classes that are concordant with the biochemical roles of the respective enzymes. We demonstrate that the analogous H 4 F pathway present in M. extorquens AM1 cannot fulfill the formaldehyde detoxification function, while a heterologously expressed pathway linked to glutathione and NAD + can successfully substitute for the H 4 MPT pathway. Additionally, null mutants were generated in genes previously thought to be essential, indicating that the H 4 MPT pathway is not absolutely required during growth on multicarbon compounds. These results define the role of the H 4 MPT pathway as the primary formaldehyde oxidation and detoxification pathway in M. extorquens AM1.

Ten novel methylotrophy genes of the facultative methylotroph Methylobacterium extorquens AM1 were identified from a transposon mutagenesis screen. One of these genes encodes a product having identity with dihydrofolate reductase (DHFR). This mutant has a C 1 -defective and methanol-sensitive phenotype that has previously only been observed for strains defective in tetrahydromethanopterin (H 4 MPT)-dependent formaldehyde oxidation. These results suggest that this gene, dmrA , may encode dihydromethanopterin reductase, an activity analogous to that of DHFR that is required for the final step of H 4 MPT biosynthesis.

Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

NADP-dependent methylene-H 4 MPT 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-H 4 F 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-H 4 MPT 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-H 4 F dehydrogenases are discussed.

Recently, it has been shown that heterotrophic methylotrophic Proteobacteria contain tetrahydrofolate (H(4)F)- and tetrahydromethanopterin (H(4)MPT)-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 H(4)MPT (specific activity: 680 U/mg). Its N-terminal amino acid sequence showed sequence identity to NAD(P)-dependent methylene H(4)MPT dehydrogenase MtdB from Methylobacterium extorquens AM1. The second dehydrogenase is specific for NADP and methylene H(4)F (specific activity: 180 U/mg) and also exhibits methenyl H(4)F cyclohydrolase activity. Via N-terminal amino acid sequencing this dehydrogenase was identified as belonging to the classical bifunctional methylene H(4)F 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 alpha-Proteobacteria. For example, FolD was not found in M. extorquens AM1, and the NADP-dependent methylene H(4)MPT dehydrogenase MtdA was present in the bacterium that also shows H(4)F activity.

Biogenic methane production is dominated by methanoarchaea of the genera Methanosarcina (Ms.) and Methanosaeta (Mt.) that grow on acetate (6). Interestingly, Methanosaeta species can use only acetate as a substrate and are therefore obligate aceticlastic methanogens. Members of this genus are of special importance for the productivity of biogas plants, especially for reactor performance and stability at low acetate concentrations. To optimize biomethanation, it is necessary to acquire a comprehensive understanding of the biochemistry of acetate-dependent methanogenesis. Energy conservation in Methanosaeta species is not well understood, and even the sequencing of the Methanosaeta thermophila genome (13) did not unravel its mechanism. Comparative genomics indicated that the core methanogenic pathway, the breakdown of acetylcoenzyme A (CoA) to methane, is obviously well conserved in Mt. thermophila. It can be concluded that acetate is activated by acetyl-CoA synthetases and the resulting acetyl-CoA serves as a substrate for a CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) that oxidizes the carbonyl group to CO 2 and reduces ferredoxin. The methyl group is first transferred to tetrahydromethanopterin and then to coenzyme M (CoM) (2

Methylotrophs grow on reduced single-carbon compounds like methylamine as the sole source of carbon and energy. In Methylobacterium extorquens AM1, the best-studied aerobic methylotroph, a periplasmic methylamine dehydrogenase that catalyzes the primary oxidation of methylamine to formaldehyde has been examined in great detail. However, recent metagenomic data from natural ecosystems are revealing the abundance and importance of lesser-known routes, such as the N -methylglutamate pathway, for methylamine oxidation. In this study, we used M. extorquens PA1, a strain that is closely related to M. extorquens AM1 but is lacking methylamine dehydrogenase, to dissect the genetics and physiology of the ecologically relevant N -methylglutamate pathway for methylamine oxidation. Phenotypic analyses of mutants with null mutations in genes encoding enzymes of the N -methylglutamate pathway suggested that γ-glutamylmethylamide synthetase is essential for growth on methylamine as a carbon source but not as a nitrogen source. Furthermore, analysis of M. extorquens PA1 mutants with defects in methylotrophy-specific dissimilatory and assimilatory modules suggested that methylamine use via the N -methylglutamate pathway requires the tetrahydromethanopterin (H 4 MPT)-dependent formaldehyde oxidation pathway but not a complete tetrahydrofolate (H 4 F)-dependent formate assimilation pathway. Additionally, we present genetic evidence that formaldehyde-activating enzyme (FAE) homologs might be involved in methylotrophy. Null mutants of FAE and homologs revealed that FAE and FAE2 influence the growth rate and FAE3 influences the yield during the growth of M. extorquens PA1 on methylamine.

The availability of thousands of sequenced genomes has revealed the diversity of biochemical solutions to similar chemical problems. Even for molecules at the heart of metabolism, such as cofactors, the pathway enzymes first discovered in model organisms like Escherichia coli or Saccharomyces cerevisiae are often not universally conserved. Tetrahydrofolate (THF) (or its close relative tetrahydromethanopterin) is a universal and essential C1-carrier that most microbes and plants synthesize de novo. The THF biosynthesis pathway and enzymes are, however, not universal and alternate solutions are found for most steps, making this pathway a challenge to annotate automatically in many genomes. Comparing THF pathway reconstructions and functional annotations of a chosen set of folate synthesis genes in specific prokaryotes revealed the strengths and weaknesses of different microbial annotation platforms. This analysis revealed that most current platforms fail in metabolic reconstruction of variant pathways. However, all the pieces are in place to quickly correct these deficiencies if the different databases were built on each other's strengths.

This chapter explores the microbiology and biochemistry of acetate conversion to methane, a key component of biomethanation. It provides a fundamental background appropriate for stimulating advances to improve the process that will ensure biomethanation among the competitive alternatives to fossil fuels. Biomethanation of organic matter in nature occurs in diverse habitats such as freshwater sediments, rice paddies, sewage digesters, the rumen, the lower intestinal tract of monogastric animals, landfills, hydrothermal vents, coastal marine sediments, and the subsurface. Methanosarcina species synthesize tetrahydrosarcinapterin (H4SPT) which serves the same function as tetrahydromethanopterin (H4MPT). The aceticlastic and CO2 reduction pathways generate primary sodium and proton gradients that are the only possible driving forces for ATP synthesis. The production of acetate from complex biomass by fermentative and acetogenic anaerobes and the subsequent conversion of acetate to methane by aceticlastic methanogens are of primary importance in the biomethanation process. Aceticlastic methanogenesis is the major factor controlling the rate and reliability of the process; thus, a comprehensive understanding of these methanogens is paramount for developing an efficient process for biomethanation of renewable and waste biomass for use as a biofuel. Although the enzymology of reactions leading from acetate to methane by Methanosarcina species is fairly well understood, there have been only a few investigations reported on the mechanism of energy conservation and regulation of gene expression. Further, global proteomic and microarray analyses have identified a host of proteins and genes in Methanosarcina species, many with unknown functions, that may be important or essential.

1 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 by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.

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.

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of α, β, γ, δ, and e subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO2, transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H4MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H4MPT. The α and e subunits are required for CO oxidation. The γ and δ subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the β subunit. The isolated β subunit contains significant amounts of nickel. When proteases truncate the β subunit, causing the CODH/ACS complex to dissociate, the amount of intact β subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the β subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the β subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C–C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.

The roles of cyclic formaldehyde oxidation via 6-phosphogluconate dehydrogenase and linear oxidation via the tetrahydromethanopterin (H4MPT)-linked pathway were assessed in an obligate methylotroph, Methylobacillus flagellatus KT, by cloning, sequencing and mutating two chromosomal regions containing genes encoding enzymes specifically involved in these pathways:6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase and methenyl H4MPT cyclohydrolase (gndA, zwf and mch). No null mutants were obtained in gndA or zwf, implying that the cyclic oxidation of formaldehyde is required for C1 metabolism in this obligate methylotroph, probably as the main energy-generating pathway. In contrast, null mutants were generated in mch, indicating that the H4MPT-linked pathway is dispensable. These mutants showed enhanced sensitivity to formaldehyde, suggesting that this pathway plays a secondary physiological role in this methylotroph. This function is in contrast to Methylobacterium extorquens AM1, in which the H4MPT-linked pathway is essential.

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.

Methylobacterium extorquens AM1 is an aerobic methylotrophic bacterium which belongs to the α-subgroup of the proteobacteria (22, 30). In this organism methanol has been proposed to be metabolized to CO2 via formaldehyde, N5,N10-methylene tetrahydrofolate (methylene H4F), N5,N10-methenyl tetrahydrofolate (methenyl H4F), N10-formyl tetrahydrofolate (formyl H4F), and formate as intermediates in reactions involving pyrroloquinolinequinone-dependent methanol dehydrogenase, NADP-dependent methylene H4F dehydrogenase, methenyl H4F cyclohydrolase, formyl H4F synthetase, and NAD-dependent formate dehydrogenase (7, 19, 21). Recently, M. extorquens AM1 was shown to contain genes thought to be unique for methanogenic archaea, namely, genes predicted to encode methenyl tetrahydromethanopterin (H4MPT) cyclohydrolase, formylmethanofuran:H4MPT formyltransferase, and formylmethanofuran dehydrogenase (6). These enzymes catalyze the first three steps of methanogenesis during the growth of methanogens on CO2 and H2 and the last three steps of CO2 formation during the growth of methanogens on methanol (15, 28). The genes in M. extorquens AM1 encoding the three methanogenic enzymes were shown by mutagenesis to be required for growth on C1 compounds, suggesting an involvement of the corresponding enzymes in formaldehyde oxidation to CO2 via N5,N10-methylene H4MPT (methylene H4MPT), N5,N10-methenyl H4MPT (methenyl H4MPT), N5-formyl H4MPT (formyl H4MPT), and formylmethanofuran as intermediates (6). The genetic evidence for this novel metabolic pathway was substantiated by the finding that cell extracts of methanol-grown M. extorquens AM1 exhibit methenyl H4MPT cyclohydrolase activity and formylmethanofuran:H4MPT formyltransferase activity and contain dephospho-H4MPT (6). H4MPT is an H4F analogue previously found only in methanogenic and sulfate-reducing archaea. Its properties are significantly different from those of H4F (Fig. ​(Fig.1).1). Thus, the redox potential of the N5,N10-methenyl H4MPT–N5,N10-methylene H4MPT couple (−390 mV) is 90 mV more negative than that of the N5,N10-methenyl H4F–N5,N10-methylene H4F couple (−300 mV) (13, 29, 37). FIG. 1 Structures of H4MPT (25, 32, 33) and H4F. Functionally, the most important difference between H4MPT and H4F is that H4MPT has an electron-donating methylene group in conjugation to N10 via the aromatic ring, whereas H4F has an electron-withdrawing carbonyl ... Further support for the novel metabolic pathway came from the finding that cell extracts of M. extorquens AM1 contain an NADP-dependent methylene H4MPT dehydrogenase activity catalyzing the formation of methenyl H4MPT from methylene H4MPT (6). The presence of an NADP-dependent methylene H4MPT dehydrogenase in M. extorquens AM1 is of special interest because the methylene H4MPT dehydrogenases in methanogenic and sulfate-reducing archaea are specific for coenzyme F420, which is a 5′ deazaflavin derivative (8). The redox potential of the F420–F420H2 couple (−360 mV) is 40 mV more negative than that of the NAD(P)+–NAD(P)H couple (−320 mV) (12, 34). Apparently, M. extorquens AM1 contains a novel enzyme combining features of both the F420-dependent methylene H4MPT dehydrogenase from methanogens and the NAD(P)-dependent methylene H4F dehydrogenase found in bacteria and eucarya. We report here on the purification and the characterization of the novel enzyme.

The methylotroph Methylobacterium extorquens AM1 oxidizes methanol and methylamine to formaldehyde and subsequently to formate, an intermediate that serves as the branch point between assimilation (formation of biomass) and dissimilation (oxidation to CO 2 ). The oxidation of formaldehyde to formate is dephosphotetrahydromethanopterin (dH 4 MPT) dependent, while the assimilation of carbon into biomass is tetrahydrofolate (H 4 F) dependent. This bacterium contains two different enzymes, MtdA and MtdB, both of which are dehydrogenases able to use methylene-dH 4 MPT, an intermediate in the oxidation of formaldehyde to formate. Unique to MtdA is a second enzymatic activity with methylene-H 4 F. Since methylene-H 4 F is the entry point into the biomass pathways, MtdA plays a key role in assimilatory metabolism. However, its role in oxidative metabolism via the dH 4 MPT-dependent pathway and its apparent inability to replace MtdB in vivo on methanol growth are not understood. Here, we have shown that an mtdB mutant is able to grow on methylamine, providing a system to study the role of MtdA. We demonstrate that the absence of MtdB results in the accumulation of methenyl-dH 4 MPT. Methenyl-dH 4 MPT is shown to be a competitive inhibitor of the reduction of methenyl-H 4 F to methylene-H 4 F catalyzed by MtdA, with an estimated K i of 10 μM. Thus, methenyl-dH 4 MPT accumulation inhibits H 4 F-dependent assimilation. Overexpression of mch in the mtdB mutant strain, predicted to reduce methenyl-dH 4 MPT accumulation, enhances growth on methylamine. Our model proposes that MtdA regulates carbon flux due to differences in its kinetic properties for methylene-dH 4 MPT and for methenyl-H 4 F during growth on single-carbon compounds.

The ftr gene encoding formylmethanofuran: tetrahydromethanopterin formyltransferase (Ftr) from Methanosarcina barkeri was cloned, sequenced, and functionally expressed in Escherichia coli. The overproduced enzyme was purified eightfold to apparent homogeneity, and its catalytic properties were determined. The primary structure and the hydropathic character of the formyltransferase from Methanosarcina barkeri were compared with those of the enzymes from Methanobacterium thermoautotrophicum, Methanothermus fervidus, and Methanopyrus kandleri. The amino acid sequence of the enzyme from Methanosarcina barkeri was 64%, 61%, and 59% identical to that of the enzyme from Methanobacterium thermoautotrophicum, Methanothermus fervidus, and Methanopyrus kandleri, respectively. A negative correlation between the hydrophobicity of the enzymes and both the growth temperature optimum and the intracellular salt concentration of the four organisms was observed. The hydrophobicity of amino acid composition was +21.6 for the enzyme from Methanosarcina barkeri (growth temperature optimum 37 degrees C, intracellular salt concentrationapproximately 0.3 M), +9.9 for the enzyme from Methanobacterium thermoautotrophicum (65 degrees C,approximately 0.7 M), -20.8 for the enzyme from Methanothermus fervidus (83 degrees C,approximately 1.0 M) and -31.4 for the enzyme from Methanopyrus kandleri (98 degrees C,1.1 M). Generally, a positive correlation between hydrophobicity and thermophilicity of enzymes and a negative correlation between hydrophobicity and halophilicity of enzymes are observed. The findings therefore indicate that the hydropathic character of the formyltransferases compared is mainly determined by the intracellular salt concentration rather than by temperature. Sequence similarities between the formyltransferases from methanogens and an open reading frame from Methylobacterium extorquens AM1 are discussed.

A protein catalyzing the tritium exchange of [5-3H]deoxyuridine monophosphate ([5-3H]dUMP) for solvent protons and the dehalogenation of 5-bromo-deoxyuridine monophosphate (Br-dUMP) has been isolated from the methanogenic archaea Methanobacterium thermoautotrophicum. These two activities are well-established side reactions of thymidylate synthase and do not require cofactors. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of the purified enzyme showed a single band with a molecular mass of 27 kDa. The suggested molecular mass of the native protein calculated from sedimentation equilibrium experiments was 33.5 kDa, indicating that the enzyme is a monomer. The pH optima were 9.0 and 7.0 for the exchange reaction and the dehalogenation, respectively. The effects of temperature, salt, reducing agent and inhibitors were determined. The apparent Km for the tritium exchange from [5-3H]dUMP was 7 microM and for the dehalogenation of Br-dUMP was 14 microM. However, thus far, the conditions for dTMP synthesis from dUMP have not yet been established. Incubation of the enzyme with dUMP, tetrahydromethanopterin, a folate analog present in methanogens, and formaldehyde did not yield dTMP. The first 30 amino acids of the amino terminus have been sequenced. However, there is no similarity with any of the thymidylate synthases. Surprisingly, the protein from M. thermoautotrophicum appears to be related to chitin synthases from several organisms.

Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H4MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabolism. These include reactions catalyzed by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase and methyl-coenzyme M reductase (MCR). The latter enzyme is central to C–H bond formation and cleavage underlying methanogenic and reverse methanogenic phenotypes. Here, we describe a set of novel tools for the detection and quantification of H4MPT-linked C1 transfer reactions mediated by uncultivated anaerobic methane-oxidizing archaea (ANME). These tools include polymerase chain reaction primers targeting ANME MCR subunit A subgroups and protein extraction methods from marine sediments compatible with high-resolution mass spectrometry for profiling community structure and functional dynamics.

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 N5,N10-methylenetetrahydromethanopterin dehydrogenase (coenzyme F420 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.

Washed invested vesicle preparations of Methanosarcina strain Go1 catalyzed the formation of methyl-CoM from formaldehyde, H2 and CoM in the presence of tetrahydromethanopterin and 2-bromoethanesulfonate. The reaction was associated with the translocation of sodium ions into the lumen of the vesicles. This translocation was abolished by the Na+ ionophore ETH 157 but it was insensitive to the addition of the uncoupler SF6847 and the Na+/H+ antiport inhibitor amiloride and, therefore, is the result of a primary Na+ pump. Since the translocation of Na+ was also observed when formaldehyde + tetrahydromethanopterin was replaced by methyl-tetrahydromethanopterin, it follows that the methyl transfer from tetrahydromethanopterin to CoM is the sodium-motive reaction. Methyl-tetrahydromethanopterin could be replaced by methyl-tetrahydrofolate.

The conversion of methyl-tetrahydromethanopterin to methylcoenzyme M in Methanosarcina barkeri is catalyzed by two enzymes: an enzyme with a bound corrinoid, which becomes methylated during the reaction and an enzyme which transfers the methyl group from this corrinoid to coenzyme M. As in the similar methyltransfer reaction in Methanobacterium thermoautotrophicum the corrinoid enzyme in M barkeri needs to be activated by H2 and ATP. ATP can be replaced by Ti(III)citrate or CO.

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110 degrees C on H2 and CO2 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 alpha-, beta- and gamma-subunits and that it contained the nickel porphinoid coenzyme F430 as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the gamma-subunit was determined. A comparison with the N-terminal sequences of the gamma-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 CO2 (apparent Vmax at 65 degrees C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran:tetrahydro-methanopterin formyltransferase, 13 U/mg; N5,N10-methylenetetrahydromethanopterin cyclohydrolase, 14U/mg; N5,N10-methenyltetrahydromethanopterin dehydrogenase (H2-forming), 33 U/mg; N5,N10-methylenetetrahydromethanopterin reductase (coenzyme F420 dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F420-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent Km values for these enzymes and the effect of salts on their activities were determined. The coenzyme F420 present in M. kandleri was identified as coenzyme F420-2 with 2-gamma-glutamyl residues.

The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis. More than 100 mutually exclusive spots were present in acetate- and methanol-grown cells. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells. Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate- and methanol-grown Methanosarcina thermophila. Levels of formyltransferase in either acetate- or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum. All other enzyme activities were significantly lower in acetate- and methanol-grown Methanosarcina thermophila.