MtxX, also known as Methanogen Marker Protein 4 (MMP4), is a member of the group of proteins conserved in archaeal methanogens called the Methanogen Marker Proteins (MMPs). Owing to this taxonomic distribution the MMPs are presumed to have roles related to methanogenesis or are evidence for an evolutionary history associated with methanogenic processes. MtxX is sequence-annotated as either a methyltransferase (EC 2.1.1.-) or a phosphate acetyl/butyryltransferase (EC 2.3.1.8/2.3.1.19). Gene synteny analysis shows mtxX is located next to other MMP genes in Methanomicrobiales, Methanotrichales, and Methanocaldococcus genomes, while in Methanobacteria and Methanococci it is positioned adjacent to undecaprenyl pyrophosphate synthase, a cell wall biosynthesis enzyme. We describe the crystal structure for MtxX from Methanothermobacter thermautotrophicus ΔH showing that it has a protein fold homologous to phosphate acetyltransferases and decarboxylating NAD(P)-dependent dehydrogenases. The MtxX structure has a conserved binding cleft which is the presumptive functional site based on crystallographic symmetry-related molecular binding interactions and structural homology.

Epimerases and dehydratases are widely studied members of the extended short-chain dehydrogenase/reductase (SDR) enzyme superfamily and are important in nucleotide sugar conversion and diversification, for example, the interconversion of uridine diphosphate (UDP)-linked glucose and galactose. Methanothermobacter thermautotrophicus contains a cluster of genes, the annotations of which indicate involvement in glycan biosynthesis such as that of cell walls or capsular polysaccharides. In particular, genes encoding UDP-glucose 4-epimerase related protein (Mth375), UDP-glucose 4-epimerase homologue (Mth380) and dTDP-glucose 4,6-dehydratase related protein (Mth373) may be involved in the biosynthesis of an unusual aminosugar in pseudomurein. In this paper, we present the structures of Mth375, an archaeal sugar epimerase/dehydratase protein (WbmF) determined to a resolution of 2.0 Å. The structure contains an N-terminal Rossmann-fold domain with bound nicotinamide adenine dinucleotide hydride (NADH) and a C-terminal catalytic domain with bound UDP. We also present the structure for Mth373 co-crystallised with uridine-5'-diphosphate-xylopyranose to a resolution of 1.96 Å as a NAD+-dependent oxidative decarboxylase (UDP-xylose synthase; EC4.1.1.35). Molecular modelling has also allowed for the identification of Mth380 as a UDP-N-acetylglucosamine 4-epimerase (WbpP; EC5.1.3.7), Mth631 as a UDP-glucose 4-epimerase (GalE; EC5.1.3.2) and Mth1789 as a classical dTDP-d-glucose 4,6-dehydratase (EC4.2.1.46). The UDP-sugar specificity of each archaeal nucleotide sugar short-chain dehydrogenase/reductase (NS-SDR) was elucidated via sequence, molecular modelling and structural analyses. Overall, these structures potentially shed light on the formation of the glycan portion of pseudomurein and capsular polysaccharide in Archaea.

Pectin is a dynamic and complex polysaccharide that forms a substantial proportion of the primary plant cell wall and middle lamella of forage ingested by grazing ruminants. Pectin methylesterases (PMEs) are enzymes that belongs to the carbohydrate esterase family 8 (CE8) and catalyze the demethylesterification of pectin, a key polysaccharide in cell walls. Here we present the crystal structure of the catalytic domain of PmeC5 that is associated with a gene from Butyrivibrio fibrisolvens D1T that encodes a large secreted pectinesterase family protein (2089 aa) determined to a resolution of 1.33 Å. Protein in silico modelling of the secreted pectinesterase confirmed the presence of an additional pectate lyase (PL9) and adhesin-like domains. The structure of PmeC5 was the characteristic right-handed parallel β-helical topology and active site residues of Asp231, Asp253, and Arg326 typical of the enzyme class. PmeC5 is a large modular enzyme that is characteristic of rumen B. fibrisolvens megaplasmids and plays a central role in degrading plant cell wall components and releasing methanol in the rumen environment. Such secreted PMEs are significant contributors to plant fiber digestion and methane production, making them attractive targets for both methane mitigation strategies and livestock productivity enhancement.

Methanosphaera spp. are methylotrophic methanogenic archaea and members of the order Methanobacteriales with few cultured representatives. Methanosphaera sp. ISO3-F5 was isolated from sheep rumen contents in New Zealand. Here, we report its complete genome, consisting of a large chromosome and a megaplasmid (GenBank accession numbers CP118753 and CP118754, respectively).

Rumen methanogens predominantly fall into two physiological groups: hydrogenotrophs which use hydrogen (H2) to reduce carbon dioxide (CO2) to methane (CH4), and methylotrophs which use H2 to reduce methanol and methylamines as substrates for methanogenesis. We used a dilution to extinction approach to isolate two hydrogenotrophic Methanocatella spp. and four cultures of methylotrophic methanogens from sheep rumen contents. Three of the methylotrophs were stable mixed cultures containing methanogens belonging to different lineages of the order Methanomassiliicoccales and one was a pure Methanosphaera culture. Methanosphaera sp. ISO3-F5 has a comparatively large genome (2.68 Mb) comprised of two replicons, a chromosome and a megaplasmid. The genome has an average G + C content of 30.5 % and encodes 2360 putative protein-coding genes. Cells of ISO3-F5 have a spherical shape, 0.6-1.2 mu m in diameter, usually occurring in pairs or loose clumps, and have no flagellum. Cells stain Gram positive, have a single thick cell wall and divide by the formation of a cross wall. The optimum temperature for growth was 39 degrees C to 42 degrees C and the optimum pH was 6.7-6.8. Acetate was required for growth, but CH4 was not produced from acetate, formate, ethanol, methylamine, or isopropanol with or without H2/CO2. Volatile fatty acids and rumen fluid were also found to enhance the growth of ISO3-F5, while coenzyme M did not. ISO3-F5 produced CH4 from methanol in the presence of H2 and the genes encoding the necessary methanogenesis pathway have been identified. Based on morphological, physiological, and genomic characteristics, ISO3-F5 is a new species of the genus Methanosphaera. Our study shows that simple isolation methods allowed us to culture diverse and significant members of the rumen methanogen community.

Methyl‐coenzyme M reductase (MCR) is a multi‐subunit (α2β2γ2) enzyme responsible for methane formation via its unique F430 cofactor. The genes responsible for producing MCR (mcrA, mcrB and mcrG) are typically colocated with two other highly conserved genes mcrC and mcrD. We present here the high‐resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin‐like domain assembled into an α + β barrel‐like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas.

Pectin is a complex polysaccharide that forms a substantial proportion of the plant's middle lamella of forage ingested by grazing ruminants. Methanol in the rumen is derived mainly from methoxy groups released from pectin by the action of pectin methylesterase (PME) and is subsequently used by rumen methylotrophic methanogens that reduce methanol to produce methane (CH4). Members of the genus Butyrivibrio are key pectin-degrading rumen bacteria that contribute to methanol formation and have important roles in fibre breakdown, protein digestion, and the biohydrogenation of fatty acids. Therefore, methanol release from pectin degradation in the rumen is a potential target for CH4 mitigation technologies. Here, we present the crystal structures of PMEs belonging to the carbohydrate esterase family 8 (CE8) from Butyrivibrio proteoclasticus and Butyrivibrio fibrisolvens, determined to a resolution of 2.30 Å. These enzymes, like other PMEs, are right-handed β-helical proteins with a well-defined catalytic site and reaction mechanisms previously defined in insect, plant, and other bacterial pectin methylesterases. Potential substrate binding domains are also defined for the enzymes.

Epimerases are widely studied members of the extended short-chain dehydrogenases/reductases (SDR) enzyme superfamily that catalyse the interconversion of UDP linked glucose and galactose [1][2].Methanothermobacter thermautotrophicus contains several annotated 4-aminosugar epimerases encoded by a cluster of genes indicating involvement in cell wall biosynthesis or capsular polysaccharide formation.We present the structure of Mth375, an archaeal GalE 4-epimerase co-crystallised with uridine-5'-diphosphate (Figure 1; EC 5.1.3.2) determined to a maximum resolution of 2.0 Å.The structure contained an Nterminal Rossmann-fold domain with bound NADH and a C-terminal catalytic domain with bound UDP.We also present the structure for Mth373 co-crystallised with uridine-5'-diphosphate-xylopyranose to a resolution of 1.96 Å (Figure 1).Molecular modelling has identified Mth380 as a UDP-N-acetylgalactosamine 4-epimerase (WbpP; EC 5.1.3.7),Mth631 another GalE 4epimerase and Mth1789 as a dtGD/dTDP-D-glucose 4,6-dehydratase (EC 4.2.1.46).The UDP-sugar specificity of the archaeal epimerase was elucidated via sequence, molecular modelling and structural analysis [3-5].Overall, these structures potentially shed light on the formation of the glycan portion of pseudomurein in archaea.Figure 1.The omit electron density around the bound molecules (Fo-Fc set to 2.5 σ) observed for Mth375 (left) with NADH and UDP and Mth373 (right) with NADH and UDX.

Agricultural methane produced by archaea in the forestomach of ruminants is a key contributor to rising levels of greenhouse gases leading to climate change. Functionalized biological polyhydroxybutyrate (PHB) nanoparticles offer a new concept for the reduction of enteric methane emissions by inhibiting rumen methanogens. Nanoparticles were functionalized in vivo with an archaeal virus lytic enzyme, PeiR, active against a range of rumen Methanobrevibacter species. The impact of functionalized nanoparticles against rumen methanogens was demonstrated in pure cultures, in rumen batch and continuous flow rumen models yielding methane reduction of up to 15% over 11 days in the most complex system. We further present evidence of biological nanoparticle fermentation in a rumen environment. Elevated levels of short-chain fatty acids essential to ruminant nutrition were recorded, giving rise to a promising new strategy combining methane mitigation with a possible increase in animal productivity.

Archaea have diverse cell wall types, yet none are identical to bacterial peptidoglycan (murein). Methanogens Methanobacteria and Methanopyrus possess cell walls of pseudomurein, a structural analogue of murein. Pseudomurein differs from murein in containing the unique archaeal sugar N-acetyltalosaminuronic acid instead of N-acetylmuramic acid, β-1,3 glycosidic bonds in place of β-1,4 bonds and only l-amino acids in the peptide cross-links. We have determined crystal structures of methanogen pseudomurein peptide ligases (termed pMurE) from Methanothermus fervidus (Mfer762) and Methanothermobacter thermautotrophicus (Mth734) that are structurally most closely related to bacterial MurE peptide ligases. The homology of the archaeal pMurE and bacterial MurE enzymes is clear both in the overall structure and at the level of each of the three domains. In addition, we identified two UDP-binding sites in Mfer762 pMurE, one at the exterior surface of the interface of the N-terminal and middle domains, and a second site at an inner surface continuous with the highly conserved interface of the three domains. Residues involved in ATP binding in MurE are conserved in pMurE, suggesting that a similar ATP-binding pocket is present at the interface of the middle and the C-terminal domains of pMurE. The presence of pMurE ligases in members of the Methanobacteriales and Methanopyrales, that are structurally related to bacterial MurE ligases, supports the idea that the biosynthetic origins of archaeal pseudomurein and bacterial peptidoglycan cell walls are evolutionarily related.

ABSTRACTBacteria near-universally contain a cell wall sacculus of murein (peptidoglycan), the synthesis of which has been intensively studied for over 50 years. In striking contrast, archaeal species possess a variety of other cell wall types, none of them closely resembling murein. Interestingly though, one type of archaeal cell wall termed pseudomurein found in the methanogen orders Methanobacteriales and Methanopyrales is a structural analogue of murein in that it contains a glycan backbone that is cross-linked by a L-amino acid peptide. Here, we present taxonomic distribution, gene cluster and phylogenetic analyses that confirm orthologues of 13 bacterial murein biosynthesis enzymes in pseudomurein-containing methanogens, most of which are distantly related to their bacterial counterparts. We also present the first structure of an archaeal pseudomurein peptide ligase from Methanothermus fervidus DSM1088 (Mfer336) to a resolution of 2.5 Å and show that it possesses a similar overall tertiary three domain structure to bacterial MurC and MurD type murein peptide ligases. Taken together the data strongly indicate that murein and pseudomurein biosynthetic pathways share a common evolutionary history.

Methane emissions from enteric fermentation in the ruminant digestive system generated by methanogenic archaea are a significant contributor to anthropogenic greenhouse gas emissions. Additionally, methane produced as an end-product of enteric fermentation is an energy loss from digested feed. To control the methane emissions from ruminants, extensive research in the last decades has been focused on developing viable enteric methane mitigation practices, particularly, using methanogen-specific inhibitors. We report here the utilization of two known inhibitors of methanogenic archaea, neomycin and chloroform, together with a recently identified inhibitor, echinomycin, to produce resistant mutants of Methanococcus maripaludis S2 and S0001. Whole-genome sequencing at high coverage (> 100-fold) was performed subsequently to investigate the potential targets of these inhibitors at the genomic level. Upon analysis of the whole-genome sequencing data, we identified mutations in a number of genetic loci pointing to potential mechanisms of inhibitor action and their underlying mechanisms of resistance.

This study was conducted to examine the effects of different levels of lovastatin on the histological and sarcoplasmic proteome profile of goat skeletal muscle. A total of 20 intact male Saanen goats were randomly assigned in equal numbers to four groups and fed a total mixed ration containing 50% rice straw, 22.8% concentrates and 27.2% of various proportions of untreated or treated palm kernel cake (PKC) to achieve the target daily intake levels of 0 (Control), 2 (Low), 4 (Medium) or 6 (High) mg lovastatin/kg BW. A histological examination discovered that the longissimus thoracis et lumborum muscle of animals from the Medium and High treatment groups showed abnormalities in terms of necrosis, degeneration, interstitial space and vacuolization. Our preliminary label-free proteomics analysis demonstrates that lovastatin supplementation induced complex modifications to the protein expression patterns of the skeletal muscle of the goat which were associated with the metabolism of carbohydrate and creatine, cell growth and development processes and other metabolic processes. The changes in these biochemical processes indicate perturbations in energy metabolism, which could play a major role in the development of myopathy. In conclusion, the present study suggests that supplementation of naturally produced lovastatin above 4 mg/kg BW could adversely affecting the health and wellbeing of treated animals.

Methane is a potent greenhouse gas (28-fold more potent than carbon dioxide) and is a significant gas contributing to global climate change. Approximately a billion tons of methane are produced each year by methanogenic archaea in ruminants. These archaea possess a number of unusual traits such as isoprenoid-based lipids, unusual cell wall chemistry and a unique energy metabolism (methanogenesis) that requires six methanogen-specific cofactors. Many of the enzymes involved in these processes have no direct analogues in the host animal. To gain insights into the fundamental biology of rumen methanogens we have determined crystal structures of key enzymes with archaeal-specific traits. Over 600 enzymes were targeted for structure determination which produced approximately 200 purified soluble enzymes for crystallographic screening. More than 50 different enzymes have produced crystals and 30 structures have been solved for individual enzymes to date. The results have helped illuminate our understanding of methane formation at this critical juncture in the world’s history.

This study investigated the carcass characteristics, physico-chemical properties, storage stability and cholesterol content of meat from goats fed with different levels of naturally-produced lovastatin used to mitigate enteric methane production. Twenty intact Saanen male goats of 5-6 months old with initial live weight of 25.8 ± 4.0 kg were randomly allotted into four dietary treatments containing 0 (Control), 2 (Low), 4 (Medium) and 6 mg (High) per kg live weight (LW) of naturally-produced lovastatin for 12 consecutive weeks. No differences were found in all the parameters measured except for full LW, hot and cold carcass weight, shear force, color and cholesterol content among the treatment groups. Aging had significant effects on all the parameters measured in this study except a* (redness) of meat. Meat samples in the Medium and High treatments were of higher lightness and yellowness, more tender and lower cholesterol levels. We conclude that, in addition to mitigate enteric methane emissions, dietary supplementation of naturally-produced lovastatin at 4 mg/kg LW could be a feasible feeding strategy to produce tender meat containing lower cholesterol.

Methane is a potent greenhouse gas and methane emissions from ruminants are widely recognized as a major contributor to global warming and climate change.One strategy for mitigating these emissions is to develop compounds that specifically inhibit the growth of archaeal methanogens.To accelerate inhibitor discovery, we targeted enzymes involved in specialized biochemical pathways of rumen methanogens (biochemically distinct from bacteria, humans and ruminant animals), including methanogenesis, cofactor biosynthesis and archaeal lipid synthesis.Over three hundred enzymes were identified as candidates for structural determination, producing in excess of one hundred soluble proteins for crystallographic screening.Over 50 different enzymes have produced crystals and 29 structures have been solved to date.A subset of these enzymes were then the focus of in vitro inhibitor screening (5 enzymes) utilising large commercial compound libraries and in silico screening (5 enzymes) to identify possible inhibitors for use in ruminants.