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. 2026 Feb 20;7:xtag011. doi: 10.1093/femsmc/xtag011

Crystal structure of methanogen MtxX (Methanogen Marker Protein MMP4) from Methanothermobacter thermautotrophicus ΔH

Andrew J Sutherland-Smith 1,✉, Vincenzo Carbone 2,✉, Wiebke Kaziur-Cegla 3, Marion Woermann 4, Linley R Schofield 5, Ron S Ronimus 6
Editor: Biswarup Mukhopadhyay
PMCID: PMC12978539  PMID: 41822306

Abstract

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.

Keywords: MtxX, MMP4, archaea, methanogen, Methanogen Marker Protein 4, Methanothermobacter thermautotrophicus ΔH

Structural analysis of the methanogen protein MtxX.

Introduction

MtxX, also known as Methanogen Marker Protein 4, is a member of the group of proteins identified as being almost unique to methanogenic archaea (Basu et al. 2011), and hence has been proposed to play a functional role in methanogen-unique pathways, being either directly or indirectly involved in methanogenesis (Gao and Gupta 2007, Leahy et al. 2010, Kaster et al. 2011, Wang et al. 2015, Borrel et al. 2019, Adler et al. 2025). mtxX is included in a set of core methanogenesis genes defined as being present in more than 90% of sequenced methanogens and less than 5% of other sequenced non-methanogenic archaea (Borrel et al. 2014). mtxX is present in class I/II methanogens, the anaerobic methanotrophic (ANME) Methanosarcinales and Syntrophoarchaeales, and the methyl-dependent hydrogenotrophic methanogens, such as Methanomassiliicoccales and Methanofastidiosales (Wang et al. 2015, Nobu et al. 2016, Borrel et al. 2019). Whole-genome transposon insertion mutagenesis in Methanococcus maripaludis S2 indicated mtxX is possibly essential (Sarmiento et al. 2013). However, mtxX is also present in some archaeal genomes that do not contain methyl coenzyme-M reductase (MCR) or mcr-like genes, for example some Archaeoglobales and Promethearchaeaceae, raising the possibility that MtxX is a remnant of a methane-associated metabolism that has diverged in function (Borrel et al. 2019, Adam et al. 2022). For Methanosarcinales mtxX is co-located in an operon with mtxA and mtxH which are predicted homologues of MtrA and MtrH (Harms and Thauer 1997, Wang et al. 2015). Cobalamin-binding MtrA, and MtrH are subunits of the membrane-bound methyltransferase (Mtr) that transfers a methyl group from methyl-H4MPT to coenzyme M, a reaction coupled to Na+ gradient generation (Harms et al. 1995). However, MtrA and MtrH are not conserved for all methanogens and are also not restricted to methanogens, being found in other archaea and bacteria (Wang et al. 2015, Adam et al. 2022).

A specific function for MtxX remains unresolved. Based on its co-localization with mtxA and mtxH in some genomes, mtxX has been annotated as a methyltransferase or methyltransferase related protein. For example, Methanothermobacter thermautotrophicus ΔH MtxX (MthMtxX) UNIPROT O26333 is annotated as an uncharacterized methyltransferase (EC 2.1.1.-). However, sequence-based homology indicates closer similarity for MtxX sequences to phosphate acetyl/butyryltransferases (phosphotransacetylases) (EC 2.3.1.8/19) (Harms and Thauer 1997, Shin 2008, Adam et al. 2022), and for many methanogen genomes mtxX is found outside any recognized methanogenesis-specific transcriptional unit, as is the case for M. thermautotrophicus ΔH. As a step in defining the role of MtxX in methanogens we have analyzed mtxX gene synteny and determined the first X-ray crystal structure of MtxX.

Materials and methods

mtxX gene synteny analysis was performed for methanogen classes Methanobacteria (49 genomes), Methanopyri (3 genomes), Methanococci (19 genomes), Methanomicrobia (98 genomes), and Methanomassiliicoccocales (7 genomes) using SyntTax (Oberto 2013), and with the MtxX protein sequence using BLASTP searches of Archaea. Organism name and taxonomic lineage were defined according to the NCBI Taxonomy database (Federhen 2012).

The MthMtxX DNA sequence (MTH_231, 1470192) was PCR subcloned into the pET151D vector for E. coli heterologous expression as a 6xHis-tagged protein. Transformed E. coli BL21(DE3) cells were grown at 37°C in 2xYT/100 μg/ml ampicillin to a cell density OD600 of 0.4–0.55 when expression was induced by the addition of isopropyl-β-D-1-thiogalactopyranoside to a concentration of 1 mM. The cultures were incubated with shaking at 25°C for overnight expression, and the cells harvested by centrifugation and frozen. For MtxX purification, the cells were thawed-refrozen for three cycles before lysozyme (final concentration 1 mg/ml) treatment to ensure lysis. DNase and RNase (final concentrations 5 μg/ml), 7 mM β-mercaptoethanol and 0.5 mM phenylmethanesulfonyl fluoride were added to the cell lysate in equilibration buffer 50 mM Tris, 10 mM imidazole, 20 mM MgCl2 and 300 mM NaCl at a ratio of 5 ml buffer per g of cell pellet. The cell lysate was filtered using a 0.22 μm Millex GP filter unit (Merck Millipore Ltd., Ireland) and applied to a Ni2+ Sepharose-6 Fast Flow column pre-rinsed in equilibration buffer. After the cell lysate was applied to the column, the unbound lysate fraction was removed by washing with equilibration buffer, followed by elution of MtxX by the addition of elution buffer (50 mM Tris, 250 mM imidazole, 20 mM MgCl2, 300 mM NaCl). The protein concentration was measured by Bradford assay and the protein purity analyzed by SDS-PAGE. MtxX was concentrated to 4 mg/ml and buffer exchanged into 20 mM MOPS pH 7, 2 mM tris(2-carboxyethyl)phosphine, 500 mM KCl and 10% (v/v) glycerol.

MtxX (filtered through a 0.22 μm membrane) was crystallized from 0.1 M Na HEPES pH 7.5, 10% (w/v) PEG 8000, 8% (v/v) ethylene glycol (Molecular Dimensions Structure Screen 2 #19) using sitting drop vapour diffusion at 21°C in a vibration free incubator. A native X-ray diffraction dataset (Table 1) was collected on a MtxX crystal soaked in mother liquor supplemented with 1 mM SrCl2 and frozen in the presence of 25% (w/v) ethylene glycol at the Australian Synchrotron beamline MX1 (Cowieson et al. 2015). The crystal was found to exhibit P21 symmetry with unit cell dimensions 56.5 72.3 65.6 Å, 90, 96.1, 90° indicating two molecules in the asymmetric unit. Butyric acid was tested as a potential MtxX ligand in crystal soaking experiments but does not show binding.

Table 1.

MthMtxX crystal, data collection and refinement parameters.

Native 0.5 M Sodium iodide soak
Space group P21 P21
Unit cell parameters:
a, b, c (Å) 56.48 72.31 65.58 56.57 72.42 65.37
α, β, γ (o) 90.00 96.12 90.00 90.00 96.10 90.00
Wavelength (Å) 0.95365 0.95372
Resolution Range (Å) 65.21–1.60 (1.63–1.60) 38.28–1.68 (1.71–1.68)
No. of observed reflections 435 174 (14 654) 409 955 (16 098)
No. of unique reflections 69 258 (3397) 59 821 (2784)
Rmeas 0.071 (1.00) 0.061 (0.75)
Rpim 0.027 (0.48) 0.023 (0.31)
CC1/2 1.00 (0.78) 1.00 (0.84)
Completeness (%) 100.0 (99.6) 99.4 (91.1)
Multiplicity 6.3 (4.3) 6.9 (5.8)
I/σ(I) 15.4 (1.0) 13.8 (1.8)
Refinement parameters:
Resolution range (Å) 65.3–1.6
Number of reflections, refined 69 236
Number of reflections, R free 3507
R factor 0.163
R free 0.185
RMSD bond lengths (Å) 0.0095
RMSD bond angles (o) 1.72
Ramachandran plot:
Favoured regions (%) 99
Allowed regions (%) 1
Outliers (%) 0
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Parameters for data in the highest resolution shell are given in parentheses

Structure determination

The MtxX structure was determined using the SIRAS method from X-ray diffraction data collected on a 0.5 M NaI soaked MtxX crystal, grown in the conditions above, at the Australian Synchrotron MX2 beamline (Aragao et al. 2018). Native and derivative diffraction data were integrated using DIALS (Winter et al. 2018) and XDS (Kabsch 2010), followed by merging and averaging the intensities with POINTLESS/AIMLESS (Evans 2011, Evans and Murshudov 2013). The MtxX crystal structure was determined using SHELX-C/D/E (Sheldrick 2010) as implemented in the CRANK2 (Skubak and Pannu 2013) pipeline accessed via CCP4 (Winn et al. 2011). SHELX located six iodide sites with occupancy greater than 0.25, resulting in a substructure determination with a combined figure of merit of 0.21, with subsequent model building yielding a correlation coefficient of 49.5. Density modification and automated building with Buccaneer (Cowtan 2006), and REFMAC5 refinement (Murshudov et al. 2011) produced a near complete structure (450 amino acids, 93%) for the two MtxX molecules in the asymmetric unit, with R/Rfree 0.23/0.26. Further cycles of combined manual rebuilding in Coot (Emsley and Cowtan 2004) and refinement using REFMAC5 improved the structure further. Local non-crystallographic symmetry restraints were used during refinement except for the final cycles of refinement. Structure stereochemistry validation with MOLPROBITY (Davis et al. 2004) shows 99% amino acids in the favoured regions of the Ramachandran plot and no outliers. Deviations from ideal stereochemistry for the MtxX structure are better than typical for the resolution of the diffraction data (Table 1). The structure contains molecule A of residues 7–242 (C-terminus), molecule B 8–235, 283 solvent molecules and one ethylene glycol molecule with R factor and Rfree of 0.163 and 0.185 (5% reflections), respectively. Overall, the electron density is well defined except for the N-terminal 40 residues of molecule B which have weaker density. The MtxX atomic coordinates and structure factors have been deposited in the RCSB protein data bank with code 9NZY. Structural figures were prepared with CCP4mg (McNicholas et al. 2011) and PyMOL (Schrödinger 2015).

Results

Gene synteny

In the Methanobacteria and Methanococci analyzed genomes mtxX is located adjacent to uppS that encodes undecaprenyl pyrophosphate synthase (Fig. 1) (e.g. M. thermautotrophicus ΔH O26334 MTH_232) a key enzyme in the biosynthesis of the undecaprenyl pyrophosphate lipid carrier for cell wall biosynthesis (Apfel et al. 1999). Recently, the cell wall biosynthesis enzymes for pseudomurein in Methanobacteria were analyzed (Subedi et al. 2021) with uppS found to be widely present in archaea. For Methanocaldococcus genomes mtxX is adjacent to mmp12 Methanogen Marker Protein 12 (Fig. 1). Within Methanomicrobia, mtxX is adjacent to mmp2 Methanogen Marker Protein 2 for Methanomicrobiales and Methanotrichales, but not for Methanosarcinales and Methanocellales (Fig. 2). mmp2 is typically associated with genes encoding other MMPs in a large cluster (MMPs-2, 3, 5, 6, 15, and 17) (Woermann 2015). The clustering of mtxX with mtxA and mtxH (Harms and Thauer 1997, Wang et al. 2015) is observed for Methanosarcina, Methanohalophilus, and Methanococcoides (Fig. 2), but not for other methanogen genomes (Fig. 1 and Fig. 2).

Figure 1.

Figure 1

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Representation of mtxX gene synteny from a subset (n = 10) of the analyzed Methanobacteria and Methanococci genomes. Annotated M. thermautotrophicus ΔH genes (left to right): hisD (light green) histinol dehydrogenase GenBank accession WBF06543.1. aspS (yellow) aspartate tRNA ligase WBF06544.1. 01025/cbiC (purple) cobalt-recorrin 8 methylmutase WBF06545.1. hemL (pink) glutamate-1-semialdehyde 2,1-aminomutase WBF06546.1. mtxX (dark blue and bold outline) methanogen markerprotein 4 WBF06549.1. uppS (red) di-trans, poly-cis-decaprenylcistransferase WBF06550.1. 01055 (green) Tat family hydrolase WBF07186.1. 01060 (beige) carboxymuconolactone decarboxylase WBF06551.1. 01075/cobN1 (grey–blue) cobaltochelatase subunit CobN WBF06554.1. Methanocaldococcus jannaschii Methanogen Marker Protein 12 mmp12 is shaded light blue. The figure was prepared from SyntTax (Oberto 2013).

Figure 2.

Figure 2

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Representation of mtxX gene synteny from a subset (n = 10) of the analyzed Methanomicrobia genomes. Methanoculleus submarinus annotated genes from left to right. 10855 (purple) universal stress protein GenBank accession UYU18191.1. 10860 (pink) archaemetzincin family Zn-dependent metalloprotease UYU18192.1. 10870 (olive) replication factor C large subunit UYU18194.1. mmp2 (10875) (pink) Methanogen Marker Protein 2 UYU18195.1. mtxX (dark blue and bold outline) methanogen marker protein 4 UYU18196.1. 10885 (green) histone family protein UYU18197.1. hisB (orange) imidazoleglycerol-phosphate dehydratase UYU18198.1. hisA (red-brown) 1-(5-phosphoribosyl)-5-[(5phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase UYU18199.1. hisG (cyan) ATP phosphoribosyltransferase UYU18200.1. 10905 (grey-blue) methionine adenosyltransferase UYU18201.1. 10910 (brown) diadenylate cyclase UYU18202.1. sucD (yellow) succinate-CoA ligase subunit alpha UYU18203.1. 10920/sucC (red) acetate-CoA ligase family protein UYU18204.1. Methanosarcinia acetivorens, Methanococcoides methylutens, and Methanohalophilus portucalensis mtxA and mtxH are shaded light grey and dark grey respectively. The figure was prepared from SyntTax (Oberto 2013).

MtxX tertiary and quaternary structure

The MtxX structure (242 amino acids) is an α/β fold comprising two subdomains (Fig. 3). The two MtxX molecules in the asymmetric unit form a dimer (Fig. 4) with 2376 Å2 of their surface area buried at the dimer interface as determined by PISA (Krissinel and Henrick 2007). Comparison of the two monomers within the dimer shows the structures to be similar with RMSD 0.53 Å for the superposition of 226 Cα atoms. MtxX contains a prominent cleft in its structure (Fig. 4) that is primarily formed between the two subdomains of a monomer, and with contributions to its structure from the non-crystallographically related monomer.

Figure 3.

Figure 3

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Cartoon representation of the MtxX tertiary structure colour-ramped N to C-terminus blue to red.

Figure 4.

Figure 4

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(A) Structure of dimeric MtxX colour-ramped N to C terminus; monomer A blue to red, and monomer B black to ivory. Residues 237'–242' of symmetry-related MtxX bound in the monomer A cleft are shown in stick representation (carbon pink, oxygen red, nitrogen blue). Selected amino acid side chains (4 Å neighbourhood) are shown with carbons coloured gold for monomer A and grey for monomer B. A bound ethylene glycol molecule (EDO) is shown. Hydrogen bonding interactions between molecules are indicated by dashed lines. (B) Close up view of symmetry-related MtxX 237–242 (pink) and neighbouring residues (gold/grey) in the cleft. (C) Electron density map (1 σ) contoured around symmetry-related 237'–242' (light blue), ethylene glycol (grey) and neighbouring residues (royal blue).

Multiple sequence alignment using MUSCLE (Edgar 2004) and Jalview (Waterhouse et al. 2009) shows that several amino acids lining the cleft, and residues at the dimer interface, are conserved (Fig. 5). This conservation of the dimer interface, the large, buried surface area and the binding clefts being formed from both monomers suggest that MtxX is a functional dimer. The cleft of one MthMtxX monomer (monomer A) is occupied by the C-terminal residues 238–242 of a crystallographic symmetry-related (−X, Y+1/2, −Z) MtxX molecule (Fig. 4). ConSurf (Landau et al. 2005) analysis of orthologous MtxX sequences mapped onto the MthMtxX structure (Fig. 6) shows that this cleft is the most conserved accessible surface and hence the most likely location for a functional site. Amino acids 238'-242' from the symmetry-related C-terminus form interactions with conserved residues within the binding cleft (amino acid numbering of the symmetry-related C-terminus is indicated by the addition of ’). R54 contacts D240’; R74, N185 and R189 contact E242’ (Fig. 4) while R216 interacts with D240’ and N238’. R128(B), from monomer B, binds D240’ and R134(B) contacts E242’. The conserved D213 (DTSR motif, Fig. 5) in the cleft interacts with N241’. S215 occupies an unusual sidechain rotamer (X1=117°) interacting with S217 in a ST-turn (Duddy et al. 2004). The cleft also contains a bound ethylene glycol molecule from the cryoprotectant solution.

Figure 5.

Figure 5

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Multiple sequence alignment of MtxX proteins from Methanothermobacter thermautotrophicus ΔH (Mth-MtxX, O26333), Methanothermobacter marburgensis (Mm-MtxX, WP_013 295 499), Methanocaldococcus jannaschii (Mj-MtxX, Q58766), Methanobrevibacter ruminantium (Mr-MtxX, WP_012 955 805), Methanopyrus kandleri (Mk-MtxX, Q8TWU1), and Methanosarcina thermophila (Mst-MtxX, WP_048 166 052), phosphate acetyltransferases (PTA) from M. thermophila (Mst-PTA, P38503, PDB: 2AF4/1QZT), Porphyromonas gingivalis (Pg-PTA, B2RK03_PORG3, PDB: 6IOX) and Treponema pallidum (Tp-PTA O83132, PDB: 8FIR), and Bdellovibrio bacteriovorus malic enzyme MaeB PTA domain (Bb-MDH, WP_080 701 372, PDB: 6ZNT), Klebsiella pneumoniae D-threonate 4-phosphate dehydrogenase (Kp-D4PDH, A0A1Y0PY16_KLEPN, PDB: 6E85), Paraburkholderia xenovorans dehydrogenase (Px-HT4PDH, RP4_PARXL, PDB: 4ATY) and Thermus thermophilus isopropylmalate dehydrogenase (Tt-IPMDH, 3_THET8, PDB: 1OSJ). The alignment is coloured in the ClustalX (Larkin et al. 2007) scheme. Conserved Mth-MtxX amino acids located in the large cleft are labelled with a red circle, or a cyan circle if they are provided by the other monomer within the dimer (e.g. R128, R134). Additional residues involved in dimerization are labelled with a blue triangle. Mth-MtxX cis-proline 89 is indicated with a green circle. The multiple sequence alignment was calculated using MUSCLE (Edgar 2004) and displayed with Jalview (Waterhouse et al. 2009).

Figure 6.

Figure 6

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MtxX dimer surface representation coloured by sequence conservation calculated by ConSurf (Landau et al. 2005) (colour ramped from purple most conserved to cyan least conserved; yellow indicates insufficient data). The view is an approximately 180° rotation about the vertical axis from the view in Fig. 4 enabling the symmetry-related MtxX C-terminus and ethylene glycol (shown in stick representation with atomic colouring carbon green, oxygen red, nitrogen blue) to be visualized in the conserved cleft. The MtxX C-terminus (right hand side) is coloured yellow owing to a nonreliable conservation score.

Comparison of the MthMtxX structure to homologous structures

Structural homology searches with Foldseek (van Kempen et al. 2024) and Dali (Holm and Laakso 2016) against the RCSB Protein Data Bank (PDB) show that MtxX is homologous to phosphate acetyl/butyryltransferase domains (PTA/PTB EC 2.3.1.8/2.3.1.19) including the non-catalytic C-terminal PTA domain of MaeB hybrid malic enzymes (Harding et al. 2021) (Table 2). There is weaker homology (by Foldseek E-value and Dali Z-score) to NAD(P)-dependent decarboxylating dehydrogenases such as PdxA D-threonate 4-phosphate dehydrogenases, cis-dihydrodiol dehydrogenases, isocitrate, and propylmalate dehydrogenases. MthMtxX is a smaller protein by between 30 and 100 amino acids compared to PTA and dehydrogenase sequences with most of the absent sequences being in subdomain 1 (Figs. 5 and 7). Analysis with Foldseek multimer (Kim et al. 2025) showed the MtxX dimerization mode is shared with homologous PTA (Fig. 7) and dehydrogenase structures, including as dimeric units within hexameric MaeB (Harding et al. 2021). Significant structural homology to methyltransferases is not detected.

Table 2.

Foldseek analysis of MtxX structural homologues.

Homologous structure E value Score Identical residues (%) Enzyme Coenzyme / substrate / ligand Organism
1YCO 7.7×10−11 304 20 Branched chain phosphotransacylase Phosphate Enterococcus faecalis V583
3U9E (3UF6) 1.7×10−10 282 15 Putative phosphate acetyl/butyryl transferase 3U9E; CoA, 3UF6; CoD Listeria monocytogenes
7T88 6.7×10−10 272 14 Phosphate acetyltransferase Escherichia coli
7VG9 8.1×10−10 262 15 Phosphotransbutyrylase (Kim and Kim 2021) Clostridium acetobutylicum
6ZNE (6ZNT) 2.6×10−9 243 18 Malic enzyme (MaeB) (Harding et al. 2021) 6ZNT; CoA Bdellovibrio bacteriovorus
1VMI 1.9×10−9 240 17 Phosphate acetyltransferase (EutD) Escherichia coli
6IOW(X) 1.4×10−8 222 14 Phosphotransacetylase (Yoshida et al. 2019) Acetyl-CoA Porphyromonas gingivalis
8FIR 3.8×10−9 213 19 Phosphoacyltransferase (PlsX); (Brautigam et al. 2023) Treponema pallidum
1VIl 3.8×10−9 206 15 Phosphoacyltransferase (PlsX) (Badger et al. 2005) Bacillus subtilis
1QZT (2AF4) 2.9×10−7 191 17 Phosphotransacetylase (Iyer et al. 2004) 2AF4; CoA Methanosarcina thermophila
6E85 4.5×10−7 175 11 D-threonate 4-phosphate dehydrogenase (PdxA); (Inniss et al. 2023) Ni, POP Klebsiella pneumoniae ST23
4ATY 4.5×10−7 170 13 Terephthalate 1,2-cis-dihydrodiol dehydrogenase (PdxA) (Bains et al. 2012) Zn Burkholderia xenovorans LB400
8GS5 6.9×10−7 163 11 Isocitrate dehydrogenase (Chen et al. 2022) Homo sapiens
5HN3 5.1×10−7 156 14 Isocitrate dehydrogenase (Shimizu et al. 2017) Mn, Isocitrate Thermococcus kodakarensis
2Y3Z (1OSJ) 3.4×10−5 127 12 Isopropylmalate dehydrogenase (Ma and Yun 2018) Thermus thermophilus
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Abbreviations; CoA coenzyme A, CoD 3ʹ-dephosphocoenzyme A, POP pyrophosphate.

Figure 7.

Figure 7

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Approximately orthogonal views of the superposition of MtxX and phosphate acetyltransferase (PTA) structures including bound coenzyme A (CoA) or acetyl-coenzyme A (acetyl-CoA). MtxX (cartoon representation), the C-terminus of symmetry-related MtxX and the ethylene glycol molecule (both stick representation) bound in the MtxX cleft are coloured violet. Methanosarcina thermophila PTA (2AF4, cartoon representation) with coenzyme A (stick representation) bound (Lawrence et al. 2006) is coloured green; the root mean square difference (RSMD) is 3.1 Å for the superposition of 213 Ca atoms with MtxX. Listeria monocytogenes PTA (3U9E) bound coenzyme A is shown in blue stick representation; RSMD is 2.4 Å for the superposition of 207 Ca with MtxX (PTA is not shown for clarity). Porphyromonas gingivalis PTA (6IOX) bound acetyl-CoA (Yoshida et al. 2019) is coloured yellow; RSMD is 3.0 Å for the superposition of 206 Ca with MtxX (PTA is not shown for clarity). CoA and acetyl-CoA are shown for a single PTA monomer. Superpositions were performed on MtxX monomer A. The upper view is approximately the same orientation as Fig. 4, the lower view is rotated ∼90° about the horizontal axis.

MtxX S215, of the DTSR conserved sequence motif, is in the binding cleft (Fig. 4) where it is equivalently positioned to a conserved serine in the active site of PTA enzymes (Fig. 5). Superposition of the Methanosarcina thermophila PTA (MstPTA) structure (Fig. 7) [PDB: 2AF4 (Lawrence et al. 2006)], aligns the catalytic S309 [also Porphyromonas gingivalis PTA (PgPTA) S311, PDB: 6IOX (Yoshida et al. 2019) and Treponema pallidum TpPTA S314, PDB: 8FIR (Brautigam et al. 2023)] onto MthMtxX S215. The hypothesis that the MthMtxX symmetry related C-terminus could be mimicking a functional MtxX binding interaction is strengthened by homology analysis showing that the bound symmetry-related D240’ occupies an equivalent site in the MthMtxX cleft (Fig. 7) as acetyl phosphate does in Bacillus subtilis PTA (Xu et al. 2005) and the coenzyme A (CoA) 3ʹ phosphate for Listeria monocytogenes PTA (LmPTA, PDB: 3U9E), and Asn241’ occupies the same location in MtxX as the CoA ribose in LmPTA and MstPTA (Lawrence et al. 2006). There is heterogeneity of CoA and acetyl-CoA binding to orthologous PTAs (Fig. 7) meaning that a detailed superposition is not possible.

Other MtxX binding cleft residues are conserved with PTA active site residues (Fig. 5), but there are also differences. MtxX R216 is the equivalent residue to the PTA essential catalytic arginine (e.g. MstPTA R310, PgPTA R312, TpPTA R315) (Lawrence et al. 2006, Brautigam et al. 2023). On the other hand, the conserved PTA aspartate (e.g. MstPTA D316, PgPTA D318, TpPTA D321) (Iyer et al. 2004) that has been proposed to act as the catalytic base in the acetyl transfer mechanism (Lawrence et al. 2006) is not conserved for MtxX (Fig. 5). The MtxX arginines R74, R128, R134 within the cleft that contact the acidic symmetry-related C-terminus are not conserved in PTAs possibly implying divergent functions.

Despite some overall structural similarity (Table 2) to dehydrogenase families, MtxX does not have histidines available to coordinate a metal ion as observed for the PdxA family (Sivaraman et al. 2003), and compared to IDH sequences MtxX lacks homologous regions of the ‘NADP-binding’ and ‘phosphorylation’ loops (Goncalves et al. 2012). For MtxX, the presence of its conserved cleft, and the fact that it is the substrate/ligand binding site in structural homologues suggests that the symmetry-related C-terminal tail binding mode observed for MtxX is mimicking a ligand interaction. The binding of the acidic C-terminal tail, the predominance of basic residues and the presence of the conserved hydrophobic amino acids F188 and L193 in the MtxX binding cleft suggests any native ligand for MtxX likely has hydrophobic as well as acidic character.

Discussion

The high-resolution crystal structure of methanogen M. thermautotrophicus ΔH MtxX has revealed a dimeric structure with a fold homologous to that of phosphate acetyltransferases and decarboxylating dehydrogenases with the identification of the potential binding/functional site cleft. MthMtxX amino acids from both monomers contribute to the two conserved clefts within the dimer. In the crystal structure, the cleft of one monomer (monomer A) binds the acidic C-terminus of a crystallographic symmetry-related MtxX molecule giving some indication of the binding properties of this site with conserved, predominantly basic, residues in the cleft interacting with the crystallographic acidic C-terminus. The MthMtxX C-terminus is not conserved for MtxX sequences from other methanogens (Fig. 5) so this binding likely does not represent a functional or regulatory interaction but does serve to highlight the conserved binding cleft and interaction mode that might mimic that of the MtxX native binding ligand. The MtxX binding site is located similarly to the active site of phosphate acetyltransferases. PTAs catalyse the reversible transfer of the acetyl group from acetyl phosphate to CoA to produce acetyl-CoA and inorganic phosphate. Within methanogens, PTAs have been annotated only for the acetoclastic Methanosarcina where they function in concert with acetate kinase producing acetyl-CoA from acetate (Fournier and Gogarten 2008, Gilmore et al. 2017); the presence of these two genes in Methanosarcina is a result of horizontal gene transfer from bacteria (Fournier and Gogarten 2008). PTA genes and activity have not been described for hydrogenotrophic CO2 reducing methanogens such as M. thermautotrophicus ΔH. For example, Methanocaldococcus jannaschii has MtxX and does not exhibit phosphate acetyltransferase, or acetyl-CoA synthetase activity (Sprott et al. 1993).

The presence of mtxX within a methyltransferase-related mtxXAH operon for some methanogen genomes had led to the annotation of MtxX as a methyltransferase, however gene synteny analysis shows that mtxX clustering with mtxA and mtxH only occurs for a subset of methanogen orders and is not typical amongst methanogen genomes. The MtxX protein structure has the highest homology to phosphate acetyltransferases and not methyltransferases. However, this structural homology does not rule out a role for MtxX in methyl group transfer processes and/or the biochemistry of specialized methanogenic archaeal processes. Further genetic and biochemical characterization of MtxX activity is required, as is the establishment of a functional assay to identify ligand molecules.

Supplementary Material

xtag011_Supplemental_File
xtag011_supplemental_file.docx (14.9KB, docx)

Acknowledgements

We appreciate support from the Australian Synchrotron in Melbourne and the New Zealand Synchrotron Group. This research was undertaken in part using the MX1 and MX2 beamlines at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector. We thank the Pastoral Greenhouse Gas Research Consortium (PGgRc) and manager Mark Aspin for their support. We thank Graeme Attwood and Nikola Palevich for helpful feedback on the manuscript.

Contributor Information

Andrew J Sutherland-Smith, School of Food Technology and Natural Sciences, Massey University, Palmerston North 4442, New Zealand.

Vincenzo Carbone, AgResearch Group, Bioeconomy Science Institute, Palmerston North 4442, New Zealand.

Wiebke Kaziur-Cegla, AgResearch Group, Bioeconomy Science Institute, Palmerston North 4442, New Zealand.

Marion Woermann, AgResearch Group, Bioeconomy Science Institute, Palmerston North 4442, New Zealand.

Linley R Schofield, AgResearch Group, Bioeconomy Science Institute, Palmerston North 4442, New Zealand.

Ron S Ronimus, AgResearch Group, Bioeconomy Science Institute, Palmerston North 4442, New Zealand.

Author contribution

AJSS, VC, LRS, WKC, MW, and RSR were responsible for the experimental design, and conducted the experimental analysis. AJSS, VC, LRS, and RSR drafted and revised the manuscript.

Conflicts of interest

None declared.

Funding

Pastoral Greenhouse Gas Research Consortium (PGgRc), New Zealand Synchrotron Group, Australian Synchrotron (ANSTO).

References

  1. Adam  PS, Kolyfetis  GE, Bornemann  TLV  et al.  Genomic remnants of ancestral methanogenesis and hydrogenotrophy in Archaea drive anaerobic carbon cycling. Sci Adv. 2022;8:eabm9651. 10.1126/sciadv.abm9651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adler  SA, Chadwick  GL, Nayak  DD.  Assembly and maturation of methyl-coenzyme M reductase in methanogenic archaea. Curr Opin Microbiol. 2025;87:102637. 10.1016/j.mib.2025.102637. [DOI] [PubMed] [Google Scholar]
  3. Apfel  CM, Takacs  B, Fountoulakis  M  et al.  Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene. J Bacteriol. 1999;181:483–92. 10.1128/JB.181.2.483-492.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aragao  D, Aishima  J, Cherukuvada  H  et al.  MX2: a high-flux undulator microfocus beamline serving both the chemical and macromolecular crystallography communities at the Australian Synchrotron. J Synchrotron Rad. 2018;25:885–91. 10.1107/S1600577518003120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Badger  J, Sauder  JM, Adams  JM  et al.  Structural analysis of a set of proteins resulting from a bacterial genomics project. Proteins. 2005;60:787–96. 10.1002/prot.20541. [DOI] [PubMed] [Google Scholar]
  6. Bains  J, Wulff  JE, Boulanger  MJ. Investigating terephthalate biodegradation: structural characterization of a putative decarboxylating cis-dihydrodiol dehydrogenase. J Mol Biol. 2012;423:284–93. 10.1016/j.jmb.2012.07.022. [DOI] [PubMed] [Google Scholar]
  7. Basu  MK, Selengut  JD, Haft  DH.  ProPhylo: partial phylogenetic profiling to guide protein family construction and assignment of biological process. BMC Bioinf. 2011;12:434. 10.1186/1471-2105-12-434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Borrel  G, Adam  PS, McKay  LJ  et al.  Wide diversity of methane and short-chain alkane metabolisms in uncultured archaea. Nat Microbiol. 2019;4:603–13. 10.1038/s41564-019-0363-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Borrel  G, Parisot  N, Harris  HM  et al.  Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine. BMC Genomics. 2014;15:679. 10.1186/1471-2164-15-679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brautigam  CA, Deka  RK, Tso  SC  et al.  Biophysical and biochemical studies support TP0094 as a phosphotransacetylase in an acetogenic energy-conservation pathway in Treponema pallidum. PLoS One. 2023;18:e0283952. 10.1371/journal.pone.0283952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen  X, Sun  P, Liu  Y  et al.  Structures of a constitutively active mutant of human IDH3 reveal new insights into the mechanisms of allosteric activation and the catalytic reaction. J Biol Chem. 2022;298:102695. 10.1016/j.jbc.2022.102695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cowieson  NP, Aragao  D, Clift  M  et al.  MX1: a bending-magnet crystallography beamline serving both chemical and macromolecular crystallography communities at the Australian Synchrotron. J Synchrotron Rad. 2015;22:187–90. 10.1107/S1600577514021717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cowtan  K.  The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D Biol Crystallogr. 2006;62:1002–11. 10.1107/S0907444906022116. [DOI] [PubMed] [Google Scholar]
  14. Davis  IW, Murray  LW, Richardson  JS  et al.  MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic Acids Res. 2004;32:W615–9. 10.1093/nar/gkh398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Duddy  WJ, Nissink  JW, Allen  FH  et al.  Mimicry by asx- and ST-turns of the four main types of beta-turn in proteins. Protein Sci. 2004;13:3051–5. 10.1110/ps.04920904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Edgar  RC.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–7. 10.1093/nar/gkh340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Emsley  P, Cowtan  K.  Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60:2126–32. 10.1107/S0907444904019158. [DOI] [PubMed] [Google Scholar]
  18. Evans  PR, Murshudov  GN.  How good are my data and what is the resolution?. Acta Crystallogr D Biol Crystallogr. 2013;69:1204–14. 10.1107/S0907444913000061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Evans  PR.  An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr. 2011;67:282–92. 10.1107/S090744491003982X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Federhen  S.  The NCBI Taxonomy database. Nucleic Acids Res. 2012;40:D136–43. 10.1093/nar/gkr1178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fournier  GP, Gogarten  JP.  Evolution of acetoclastic methanogenesis in Methanosarcina via horizontal gene transfer from cellulolytic Clostridia. J Bacteriol. 2008;190:1124–7. 10.1128/JB.01382-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gao  B, Gupta  RS.  Phylogenomic analysis of proteins that are distinctive of Archaea and its main subgroups and the origin of methanogenesis. BMC Genomics. 2007;8:86. 10.1186/1471-2164-8-86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gilmore  SP, Henske  JK, Sexton  JA  et al.  Genomic analysis of methanogenic archaea reveals a shift towards energy conservation. BMC Genomics. 2017;18:639. 10.1186/s12864-017-4036-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Goncalves  S, Miller  SP, Carrondo  MA  et al.  Induced fit and the catalytic mechanism of isocitrate dehydrogenase. Biochemistry. 2012;51:7098–115. 10.1021/bi300483w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Harding  CJ, Cadby  IT, Moynihan  PJ  et al.  A rotary mechanism for allostery in bacterial hybrid malic enzymes. Nat Commun. 2021;12:1228. 10.1038/s41467-021-21528-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Harms  U, Thauer  RK.  Identification of the active site histidine in the corrinoid protein MtrA of the energy-conserving methyltransferase complex from Methanobacterium thermoautotrophicum. Eur J Biochem. 1997;250:783–8. 10.1111/j.1432-1033.1997.00783.x. [DOI] [PubMed] [Google Scholar]
  27. Harms  U, Weiss  DS, Gartner  P  et al.  The energy conserving N5-methyltetrahydromethanopterin:coenzyme M methyltransferase complex from Methanobacterium thermoautotrophicum is composed of eight different subunits. Eur J Biochem. 1995;228:640–8. 10.1111/j.1432-1033.1995.0640m.x. [DOI] [PubMed] [Google Scholar]
  28. Holm  L, Laakso  LM.  Dali server update. Nucleic Acids Res. 2016;44:W351–5. 10.1093/nar/gkw357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Inniss  NL, Kochan  TJ, Minasov  G  et al.  A structural systems biology approach to high-risk CG23 Klebsiella pneumoniae. Microbiol Resour Announc. 2023;12:e0101322. 10.1128/mra.01013-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Iyer  PP, Lawrence  SH, Luther  KB  et al.  Crystal structure of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila. Structure. 2004;12:559–67. 10.1016/j.str.2004.03.007. [DOI] [PubMed] [Google Scholar]
  31. Kabsch  W.  XDS. Acta Crystallogr D Biol Crystallogr. 2010;66:125–32. 10.1107/S0907444909047337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kaster  AK, Goenrich  M, Seedorf  H  et al.  More than 200 genes required for methane formation from H2 and CO2 and energy conservation are present in Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus. Archaea. 2011;2011:1. 10.1155/2011/973848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kim  S, Kim  KJ. Crystal structure and molecular mechanism of phosphotransbutyrylase from Clostridium acetobutylicum. J Microbiol Biotechnol. 2021;31:1393–400. 10.4014/jmb.2109.09036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kim  W, Mirdita  M, Levy Karin  E  et al.  Rapid and sensitive protein complex alignment with Foldseek-Multimer. Nat Methods. 2025;22:469–72. 10.1038/s41592-025-02593-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Krissinel  E, Henrick  K.  Inference of macromolecular assemblies from crystalline state. J Mol Biol. 2007;372:774–97. 10.1016/j.jmb.2007.05.022. [DOI] [PubMed] [Google Scholar]
  36. Landau  M, Mayrose  I, Rosenberg  Y  et al.  ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 2005;33:W299–302. 10.1093/nar/gki370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Larkin  MA, Blackshields  G, Brown  NP  et al.  Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–8. 10.1093/bioinformatics/btm404. [DOI] [PubMed] [Google Scholar]
  38. Lawrence  SH, Luther  KB, Schindelin  H  et al.  Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila. J Bacteriol. 2006;188:1143–54. 10.1128/JB.188.3.1143-1154.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Leahy  SC, Kelly  WJ, Altermann  E  et al.  The genome sequence of the rumen methanogen Methanobrevibacter ruminantium reveals new possibilities for controlling ruminant methane emissions. PLoS One. 2010;5:e8926. 10.1371/journal.pone.0008926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ma  R, Yun  CH. Crystal structures of pan-IDH inhibitor AG-881 in complex with mutant human IDH1 and IDH2. Biochem Biophys Res Commun. 2018;503:2912–7. 10.1016/j.bbrc.2018.08.068. [DOI] [PubMed] [Google Scholar]
  41. McNicholas  S, Potterton  E, Wilson  KS  et al.  Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr D Biol Crystallogr. 2011;67:386–94. 10.1107/S0907444911007281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Murshudov  GN, Skubak  P, Lebedev  AA  et al.  REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr. 2011;67:355–67. 10.1107/S0907444911001314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nobu  MK, Narihiro  T, Kuroda  K  et al.  Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methyl-reducing methanogen. ISME J. 2016;10:2478–87. 10.1038/ismej.2016.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Oberto  J.  SyntTax: a web server linking synteny to prokaryotic taxonomy. BMC Bioinf. 2013;14:4. 10.1186/1471-2105-14-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sarmiento  F, Mrazek  J, Whitman  WB.  Genome-scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis. Proc Natl Acad Sci USA. 2013;110:4726–31. 10.1073/pnas.1220225110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schrödinger  L.  The PyMOL molecular graphics system version 3.1. 2015.
  47. Sheldrick  GM.  Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr D Biol Crystallogr. 2010;66:479–85. 10.1107/S0907444909038360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shimizu  T, Yin  L, Yoshida  A  et al.  Structure and function of an ancestral-type beta-decarboxylating dehydrogenase from Thermococcus kodakarensis. Biochem J. 2017;474:105–22. 10.1042/BCJ20160699. [DOI] [PubMed] [Google Scholar]
  49. Shin  DH.  Preliminary structural studies on the MtxX protein from Methanococcus jannaschii. Acta Crystallogr F Struct Biol Cryst Commun. 2008;64:300–3. 10.1107/S1744309108007033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sivaraman  J, Li  Y, Banks  J  et al.  Crystal structure of Escherichia coli PdxA, an enzyme involved in the pyridoxal phosphate biosynthesis pathway. J Biol Chem. 2003;278:43682–90. 10.1074/jbc.M306344200. [DOI] [PubMed] [Google Scholar]
  51. Skubak  P, Pannu  NS.  Automatic protein structure solution from weak X-ray data. Nat Commun. 2013;4:2777. 10.1038/ncomms3777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sprott  GD, Ekiel  I, Patel  GB.  Metabolic pathways in Methanococcus jannaschii and other methanogenic bacteria. Appl Environ Microb. 1993;59:1092–8. 10.1128/aem.59.4.1092-1098.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Subedi  BP, Martin  WF, Carbone  V  et al.  Archaeal pseudomurein and bacterial murein cell wall biosynthesis share a common evolutionary ancestry. FEMS Microbes. 2021;2:xtab012. 10.1093/femsmc/xtab012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. van Kempen  M, Kim  SS, Tumescheit  C  et al.  Fast and accurate protein structure search with Foldseek. Nat Biotechnol. 2024;42:243–6. 10.1038/s41587-023-01773-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wang  S, Chen  Y, Cao  Q  et al.  Long-lasting gene conversion shapes the convergent evolution of the critical methanogenesis genes. G3. 2015;5:2475–86. 10.1534/g3.115.020180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Waterhouse  AM, Procter  JB, Martin  DM  et al.  Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25:1189–91. 10.1093/bioinformatics/btp033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Winn  MD, Ballard  CC, Cowtan  KD  et al.  Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67:235–42. 10.1107/S0907444910045749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Winter  G, Waterman  DG, Parkhurst  JM  et al.  DIALS: implementation and evaluation of a new integration package. Acta Crystallogr D Struct Biol. 2018;74:85–97. 10.1107/S2059798317017235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Woermann  M.  Investigation of Methanogenesis Marker Proteins: implications for Environmental Processes. MSc Thesis. University of Duisburg-Essen, 2015. [Google Scholar]
  60. Xu  QS, Jancarik  J, Lou  Y  et al.  Crystal structures of a phosphotransacetylase from Bacillus subtilis and its complex with acetyl phosphate. J Struct Funct Genomics. 2005;6:269–79. 10.1007/s10969-005-9001-9. [DOI] [PubMed] [Google Scholar]
  61. Yoshida  Y, Sato  M, Nonaka  T  et al.  Characterization of the phosphotransacetylase-acetate kinase pathway for ATP production in Porphyromonas gingivalis. Journal of Oral Microbiology. 2019;11:1588086. 10.1080/20002297.2019.1588086. [DOI] [PMC free article] [PubMed] [Google Scholar]

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