Browsing by Author "Schofield LR"

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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.
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.
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.
Structural characterisation of nucleotide sugar short-chain dehydrogenases/reductases from the thermophilic pseudomurein-containing methanogen Methanothermobacter thermautotrophicus ΔH
(John Wiley and Sons Ltd on behalf of Federation of European Biochemical Societies, 2025-09-03) Carbone V; Schofield LR; Edwards PJB; Sutherland-Smith AJ; Ronimus RS
Epimerases and dehydratases are widely studied members of the extendedshort-chain dehydrogenase/reductase (SDR) enzyme superfamily and areimportant in nucleotide sugar conversion and diversiﬁcation, for example,the interconversion of uridine diphosphate (UDP)-linked glucose andgalactose. Methanothermobacter thermautotrophicus contains a cluster ofgenes, the annotations of which indicate involvement in glycan biosynthesissuch as that of cell walls or capsular polysaccharides. In particular, genesencoding UDP-glucose 4-epimerase related protein (Mth375), UDP-glucose4-epimerase homologue (Mth380) and dTDP-glucose 4,6-dehydrataserelated protein (Mth373) may be involved in the biosynthesis of an unusualaminosugar in pseudomurein. In this paper, we present the structures ofMth375, an archaeal sugar epimerase/dehydratase protein (WbmF) deter-mined to a resolution of 2.0 A. The structure contains an N-terminalRossmann-fold domain with bound nicotinamide adenine dinucleotidehydride (NADH) and a C-terminal catalytic domain with bound UDP. Wealso present the structure for Mth373 co-crystallised with uridine-50-diphosphate-xylopyranose to a resolution of 1.96 A as a NAD+-dependentoxidative decarboxylase (UDP-xylose synthase; EC4.1.1.35). Molecularmodelling has also allowed for the identiﬁcation of Mth380 as aUDP-N-acetylglucosamine 4-epimerase (WbpP; EC5.1.3.7), Mth631 as aUDP-glucose 4-epimerase (GalE; EC5.1.3.2) and Mth1789 as a classicaldTDP-D-glucose 4,6-dehydratase (EC4.2.1.46). The UDP–sugar speciﬁcityof each archaeal nucleotide sugar short-chain dehydrogenase/reductase.