Methane is the second most important greenhouse gas on earth. It is produced by methanogenic archaea, which play an important role in the global carbon cycle. Three main methanogenesis pathways are ...known: in the hydrogenotrophic pathway H
2
and carbon dioxide are used for methane production, whereas in the methylotrophic pathway small methylated carbon compounds like methanol and methylated amines are used. In the aceticlastic pathway, acetate is disproportionated to methane and carbon dioxide. However, next to these conventional substrates, further methanogenic substrates and pathways have been discovered. Several phylogenetically distinct methanogenic lineages (
Methanosphaera
,
Methanimicrococcus
,
Methanomassiliicoccus
,
Methanonatronarchaeum
) have evolved hydrogen-dependent methylotrophic methanogenesis without the ability to perform either hydrogenotrophic or methylotrophic methanogenesis. Genome analysis of the deep branching
Methanonatronarchaeum
revealed an interesting membrane-bound hydrogenase complex affiliated with the hardly described class 4 g of multisubunit hydrogenases possibly providing reducing equivalents for anabolism. Furthermore, methylated sulfur compounds such as methanethiol, dimethyl sulfide, and methylmercaptopropionate were described to be converted into adapted methylotrophic methanogenesis pathways of
Methanosarcinales
strains. Moreover, recently it has been shown that the methanogen
Methermicoccus shengliensis
can use methoxylated aromatic compounds in methanogenesis. Also, tertiary amines like choline (
N
,
N
,
N
-trimethylethanolamine) or betaine (
N
,
N
,
N
-trimethylglycine) have been described as substrates for methane production in
Methanococcoides
and
Methanolobus
strains. This review article will provide in-depth information on genome-guided metabolic reconstructions, physiology, and biochemistry of these unusual methanogenesis pathways.
Key points
•
Newly discovered methanogenic substrates and pathways are reviewed for the first time.
• The review provides an in-depth analysis of unusual methanogenesis pathways.
• The hydrogenase complex of the deep branching Methanonatronarchaeum is analyzed.
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 (H
), 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 C
-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 (F
H
re-oxidation) as found for methylotrophic methanogenesis.
Methane (CH
) is a potent greenhouse gas significantly contributing to the climate warming we are currently facing. Microorganisms play an important role in the global CH
cycle that is controlled by ...the balance between anaerobic production via methanogenesis and CH
removal via methanotrophic oxidation. Research in recent decades advanced our understanding of CH
oxidation, which until 1976 was believed to be a strictly aerobic process. Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is now known to be an important sink of CH
in marine ecosystems. Furthermore, in 2006 it was discovered that anaerobic CH
oxidation can also be coupled to nitrate reduction (N-DAMO), demonstrating that AOM may be much more versatile than previously thought and linked to other electron acceptors. In consequence, an increasing number of studies in recent years showed or suggested that alternative electron acceptors can be used in the AOM process including Fe
, Mn
, As
, Cr
, Se
, Sb
, V
, and Br
. In addition, humic substances as well as biochar and perchlorate (ClO
) were suggested to mediate AOM. Anaerobic methanotrophic archaea, the so-called ANME archaea, are key players in the AOM process, yet we are still lacking deeper understanding of their metabolism, electron acceptor preferences and their interaction with other microbial community members. It is still not clear whether ANME archaea can oxidize CH
and reduce metallic electron acceptors independently or via electron transfer to syntrophic partners, interspecies electron transfer, nanowires or conductive pili. Therefore, the aim of this review is to summarize and discuss the current state of knowledge about ANME archaea, focusing on their physiology, metabolic flexibility and potential to use various electron acceptors.
Massive efforts are invested in developing innovative CO2‐sequestration strategies to counter climate change and transform CO2 into higher‐value products. CO2‐capture by reduction is a chemical ...challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2, it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2‐reducing‐and‐fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2‐fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.
The tetrathionate/thiosulfate interconversion is a two-electron process: S4O6 2– + 2 e– ↔ 2 S2O3 2–. Both transformations can support bacterial growth since S2O3 2– provides an energy source, while ...S4O6 2– serves as respiratory electron acceptor. Interest in the corresponding S2O3 2– oxidation also arises from its widespread use in volumetric analysis of oxidizing agents and bleach neutralization during water treatment. Here we report protein film electrochemistry that defines the reduction potential of the S4O6 2–/S2O3 2– couple. The relevant interconversion is not reversible at inert electrodes. However, facile reduction of S4O6 2– to S2O3 2– and the reverse reaction are catalyzed by enzymes of the thiosulfate dehydrogenase, TsdA, family adsorbed on graphite electrodes. Zero-current potentials measured with different enzymes, at three pH values, and multiple S4O6 2– and S2O3 2– concentrations together with the relevant Nernst equation resolved the tetrathionate/thiosulfate reduction potential as +198 ± 4 mV versus SHE. This potential lies in the ∼250 mV window encompassing previously reported values calculated from parameters including the free energy of formation. However, the value is considerably more positive than widely used in discussions of bacterial bioenergetics. As a consequence anaerobic respiration by tetrathionate reduction is likely to be more prevalent than presently thought in tetrathionate-containing environments such as marine sediments and the human gut.
The anaerobic oxidation of methane (AOM) is a microbial process present in marine and freshwater environments. AOM is important for reducing the emission of the second most important greenhouse gas ...methane. In marine environments anaerobic methanotrophic archaea (ANME) are involved in sulfate-reducing AOM. In contrast, Ca. Methanoperedens of the ANME-2d cluster carries out nitrate AOM in freshwater ecosystems. Despite the importance of those organisms for AOM in non-marine environments little is known about their lipid composition or carbon sources. To close this gap, we analysed the lipid composition of ANME-2d archaea and found that they mainly synthesise archaeol and hydroxyarchaeol as well as different (hydroxy-) glycerol dialkyl glycerol tetraethers, albeit in much lower amounts. Abundant lipid headgroups were dihexose, monomethyl-phosphatidyl ethanolamine and phosphatidyl hexose. Moreover, a monopentose was detected as a lipid headgroup that is rare among microorganisms. Batch incubations with 13C labelled bicarbonate and methane showed that methane is the main carbon source of ANME-2d archaea varying from ANME-1 archaea that primarily assimilate dissolved inorganic carbon (DIC). ANME-2d archaea also assimilate DIC, but to a lower extent than methane. The lipid characterisation and analysis of the carbon source of Ca. Methanoperedens facilitates distinction between ANME-2d and other ANMEs.
Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these ...enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO
, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (
of 8.4 μmol mg
min
and
of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (
of 0.4 μmol mg
min
and
of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar
values for both directions (
of 0.9 μmol mg
min
versus
of 0.3 μmol mg
min
). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with
C
-labeled acetate produced 3 μmol of
Cmethane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g dry weight
min
). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production.
Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the
activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.
The enzymes of the thiosulfate dehydrogenase (TsdA) family are wide-spread diheme c-type cytochromes. Here, redox carriers were studied mediating the flow of electrons arising from thiosulfate ...oxidation into respiratory or photosynthetic electron chains. In a number of organisms, including Thiomonas intermedia and Sideroxydans lithotrophicus, the tsdA gene is immediately preceded by tsdB encoding for another diheme cytochrome. Spectrophotometric experiments in combination with enzymatic assays in solution showed that TsdB acts as an effective electron acceptor of TsdA in vitro when TsdA and TsdB originate from the same source organism. Although TsdA covers a range from −300 to +150 mV, TsdB is redox active between −100 and +300 mV, thus enabling electron transfer between these hemoproteins. The three-dimensional structure of the TsdB-TsdA fusion protein from the purple sulfur bacterium Marichromatium purpuratum was solved by X-ray crystallography to 2.75 Å resolution providing insights into internal electron transfer. In the oxidized state, this tetraheme cytochrome c contains three hemes with axial His/Met ligation, whereas heme 3 exhibits the His/Cys coordination typical for TsdA active sites. Interestingly, thiosulfate is covalently bound to Cys330 on heme 3. In several bacteria, including Allochromatium vinosum, TsdB is not present, precluding a general and essential role for electron flow. Both AvTsdA and the MpTsdBA fusion react efficiently in vitro with high potential iron-sulfur protein from A. vinosum (Em +350 mV). High potential iron-sulfur protein not only acts as direct electron donor to the reaction center in anoxygenic phototrophs but can also be involved in aerobic respiratory chains.
Summary
Methoxylated aromatic compounds (MACs) are important components of lignin found in significant amounts in the subsurface. Recently, the methanogenic archaeon Methermicoccus shengliensis was ...shown to be able to use a variety of MACs during methoxydotrophic growth. After a molecular survey, we found that the hyperthermophilic non‐methanogenic archaeon Archaeoglobus fulgidus also encodes genes for a bacterial‐like demethoxylation system. In this study, we performed growth and metabolite analysis, and used transcriptomics to investigate the response of A. fulgidus during growth on MACs in comparison to growth on lactate. We observed that A. fulgidus converts MACs to their hydroxylated derivatives with CO2 as the main product and sulfate as electron acceptor. Furthermore, we could show that MACs improve the growth of A. fulgidus in the presence of organic substrates such as lactate. We also found evidence that other archaea such as Bathyarchaeota, Lokiarchaeota, Verstraetearchaeota, Korarchaeota, Helarchaeota and Nezhaarchaeota encode a demethoxylation system. In summary, we here describe the first non‐methanogenic archaeon with the ability to grow on MACs indicating that methoxydotrophic archaea might play a so far underestimated role in the global carbon cycle.
Thiosulfate dehydrogenases are bacterial cytochromes that contribute to the oxidation of inorganic sulfur. The active sites of these enzymes contain low-spin c-type heme with Cys–/His axial ligation. ...However, the reduction potentials of these hemes are several hundred mV more negative than that of the thiosulfate/tetrathionate couple (E m, +198 mV), making it difficult to rationalize the thiosulfate oxidizing capability. Here, we describe the reaction of Campylobacter jejuni thiosulfate dehydrogenase (TsdA) with sulfite, an analogue of thiosulfate. The reaction leads to stoichiometric conversion of the active site Cys to cysteinyl sulfonate (Cα-CH2-S-SO3 –) such that the protein exists in a form closely resembling a proposed intermediate in the pathway for thiosulfate oxidation that carries a cysteinyl thiosulfate (Cα-CH2-S-SSO3 –). The active site heme in the stable sulfonated protein displays an E m approximately 200 mV more positive than the Cys–/His-ligated state. This can explain the thiosulfate oxidizing activity of the enzyme and allows us to propose a catalytic mechanism for thiosulfate oxidation. Substrate-driven release of the Cys heme ligand allows that side chain to provide the site of substrate binding and redox transformation; the neighboring heme then simply provides a site for electron relay to an appropriate partner. This chemistry is distinct from that displayed by the Cys-ligated hemes found in gas-sensing hemoproteins and in enzymes such as the cytochromes P450. Thus, a further class of thiolate-ligated hemes is proposed, as exemplified by the TsdA centers that have evolved to catalyze the controlled redox transformations of inorganic oxo anions of sulfur.
PDF
