Browsing by Author "Ronimus RS"

Now showing 1 - 7 of 7
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    Archaeal pseudomurein and bacterial murein cell wall biosynthesis share a common evolutionary ancestry
    (FEMS Oxford University Press, 24/08/2021) Subedi B; Martin WF; Carbone V; Duin EC; Cronin B; Sauter J; Schofield LR; Sutherland-Smith A; Ronimus RS
    Bacteria 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 A 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.
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    Complete genome sequence of Methanosphaera sp. ISO3-F5, a rumen methylotrophic methanogen.
    (American Society for Microbiology, 2024-04-11) Palevich N; Jeyanathan J; Reilly K; Palevich FP; Maclean PH; Li D; Altermann E; Kelly WJ; Leahy SC; Attwood GT; Ronimus RS; Henderson G; Janssen PH; Stedman KM
    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).
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    Inhibition of Rumen Methanogens by a Novel Archaeal Lytic Enzyme Displayed on Tailored Bionanoparticles.
    (Frontiers Media S.A., 2018-10-09) Altermann E; Schofield LR; Ronimus RS; Beattie AK; Reilly K; Neubauer P
    Methane is a potent greenhouse gas, 25 times more efficient at trapping heat than carbon dioxide. Ruminant methane emissions contribute almost 30% to anthropogenic sources of global atmospheric methane levels and a reduction in methane emissions would significantly contribute to slowing global temperature rises. Here we demonstrate the use of a lytic enyzme, PeiR, from a methanogen virus that infects Methanobrevibacter ruminantium M1 as an effective agent inhibiting a range of rumen methanogen strains in pure culture. We determined the substrate specificity of soluble PeiR and demonstrated that the enzyme is capable of hydrolysing the pseudomurein cell walls of methanogens. Subsequently, peiR was fused to the polyhydroxyalkanoate (PHA) synthase gene phaC and displayed on the surface of PHA bionanoparticles (BNPs) expressed in Eschericia coli via one-step biosynthesis. These tailored BNPs were capable of lysing not only the original methanogen host strain, but a wide range of other rumen methanogen strains in vitro. Methane production was reduced by up to 97% for 5 days post-inoculation in the in vitro assay. We propose that tailored BNPs carrying anti-methanogen enzymes represent a new class of methane inhibitors. Tailored BNPs can be rapidly developed and may be able to modulate the methanogen community in vivo with the aim to lower ruminant methane emissions without impacting animal productivity.
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    Structural characterisation of methanogen pseudomurein cell wall peptide ligases homologous to bacterial MurE/F murein peptide ligases.
    (2022-09) Subedi BP; Schofield LR; Carbone V; Wolf M; Martin WF; Ronimus RS; Sutherland-Smith AJ
    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.
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    Structural determination of archaeal UDP-N-acetylglucosamine 4-epimerase from Methanobrevibacter ruminantium M1 in complex with the bacterial cell wall intermediate UDP-N-acetylmuramic acid
    (2/12/2018) Carbone V; Schofield L; Sang C; Sutherland-Smith A; Ronimus RS
    The crystal structure of UDP-N-acetylglucosamine 4-epimerase (UDP-GlcNAc 4-epimerase; WbpP; EC 5.1.3.7), from the archaeal methanogen Methanobrevibacter ruminantium strain M1, was determined to a resolution of 1.65 Å. The structure, with a single monomer in the crystallographic asymmetric unit, contained a conserved N-terminal Rossmann fold for nucleotide binding and an active site positioned in the C-terminus. UDP-GlcNAc 4-epimerase is a member of the short-chain dehydrogenase/reductase superfamily, sharing sequence motifs and structural elements characteristic of this family of oxidoreductases and bacterial 4-epimerases. The protein was co-crystallized with coenzyme NADH and UDP-N-acetylmuramic acid, the latter an unintended inclusion and well known product of the bacterial enzyme MurB and a critical intermediate for bacterial cell wall synthesis. This is a non-native UDP sugar amongst archaea and was most likely incorporated from the Eschericha coli expression host during purification of the recombinant enzyme.
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    Tailored Nanoparticles With the Potential to Reduce Ruminant Methane Emissions.
    (Frontiers Media S.A., 2022-03-11) Altermann E; Reilly K; Young W; Ronimus RS; Muetzel S; Tsapekos P
    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.
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    The Structural and Functional Characterization of Mammalian ADP-dependent Glucokinase.
    (19/02/2016) Richter JP; Goroncy AK; Ronimus RS; Sutherland-Smith AJ
    The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.