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Rudolf K. Thauer - One of the best experts on this subject based on the ideXlab platform.
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anaerobic oxidation of methane with sulfate on the reversibility of the reactions that are catalyzed by enzymes also involved in methanogenesis from co2
Current Opinion in Microbiology, 2011Co-Authors: Rudolf K. ThauerAbstract:Anaerobic oxidation of methane (AOM) with sulfate is apparently catalyzed by an association of methanotrophic archaea (ANME) and sulfate-reducing bacteria. In many habitats, the free energy change (ΔG) available through this process is only -20 kJ/mol and therefore AOM with sulfate reduction generating life-supporting ATP is predicted to operate near thermodynamic equilibrium (ΔG=0 kJ/mol). On the basis of meta-genome sequencing and enzyme studies, it has been proposed that AOM in ANME is catalyzed by the same enzymes that catalyze CO2 reduction to CH4 in methanogenic archaea. Here, this proposal is reviewed and evaluated in terms of the process thermodynamics, kinetics, and enzyme reversibilities. Currently, there is no evidence for the presence of the gene that encodes methylene-Tetrahydromethanopterin reductase in ANME, one of the central enzymes in the CO2 to CH4 pathway. However, all of the remaining enzymes do appear to be present and, with the exception of a coenzyme M-S-S-coenzyme B heterodisulfide reductase, all of these enzymes have been confirmed to catalyze reversible reactions.
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the structure of formylmethanofuran Tetrahydromethanopterin formyltransferase in complex with its coenzymes
Journal of Molecular Biology, 2006Co-Authors: Priyamvada Acharya, Rudolf K. Thauer, Ulrich Ermler, Eberhard Warkentin, Seigo ShimaAbstract:Formylmethanofuran:Tetrahydromethanopterin formyltransferase is an essential enzyme in the one-carbon metabolism of methanogenic and sulfate-reducing archaea and of methylotrophic bacteria. The enzyme, which is devoid of a prosthetic group, catalyzes the reversible formyl transfer between the two substrates coenzyme methanofuran and coenzyme Tetrahydromethanopterin (H 4 MPT) in a ternary complex catalytic mechanism. The structure of the formyltransferase without its coenzymes has been determined earlier. We report here the structure of the enzyme in complex with both coenzymes at a resolution of 2.0 A. Methanofuran, characterized for the first time in an enzyme structure, is embedded in an elongated cleft at the homodimer interface and fixed by multiple hydrophobic interactions. In contrast, Tetrahydromethanopterin is only weakly bound in a shallow and wide cleft that provides two binding sites. It is assumed that the binding of the bulky coenzymes induces conformational changes of the polypeptide in the range of 3 A that close the H 4 MPT binding cleft and position the reactive groups of both substrates optimally for the reaction. The key residue for substrate binding and catalysis is the strictly conserved Glu245. Glu245, embedded in a hydrophobic region and completely buried upon Tetrahydromethanopterin binding, is presumably protonated prior to the reaction and is thus able to stabilize the tetrahedral oxyanion intermediate generated by the nucleophilic attack of the N 5 atom of Tetrahydromethanopterin onto the formyl carbon atom of formylmethanofuran.
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Formaldehyde activating enzyme (Fae) and hexulose-6-phosphate synthase (Hps) in Methanosarcina barkeri: a possible function in ribose-5-phosphate biosynthesis
Archives of Microbiology, 2005Co-Authors: Meike Goenrich, Rudolf K. Thauer, Hiroya Yurimoto, Nobuo KatoAbstract:Formaldehyde activating enzyme (Fae) was first discovered in methylotrophic bacteria, where it is involved in the oxidation of methanol to CO_2 and in formaldehyde detoxification. The 18 kDa protein catalyzes the condensation of formaldehyde with Tetrahydromethanopterin (H_4MPT) to methylene-H_4MPT. We describe here that Fae is also present and functional in the methanogenic archaeon Methanosarcina barkeri . The faeA homologue in the genome of M. barkeri was heterologously expressed in Escherichia coli and the overproduced purified protein shown to actively catalyze the condensation reaction: apparent V _max=13 U/mg protein (1 U=μmol/min); apparent Km for H_4MPT=30 μM; apparent Km for formaldehyde=0.1 mM. By Western blot analysis the concentration of Fae in cell extracts of M. barkeri was determined to be in the order of 0.1% of the soluble cell proteins. Besides the faeA gene the genome of M. barkeri harbors a second gene, faeB-hpsB , which is shown to code for a 42 kDa protein with both Fae activity (3.6 U/mg) and hexulose-6-phosphate synthase (Hps) activity (4.4 U/mg). The results support the recent proposal that in methanogenic archaea Fae and Hps could have a function in ribose phosphate synthesis.
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how an enzyme binds the c1 carrier Tetrahydromethanopterin structure of the Tetrahydromethanopterin dependent formaldehyde activating enzyme fae from methylobacterium extorquens am1
Journal of Biological Chemistry, 2005Co-Authors: Priyamvada Acharya, Christoph H. Hagemeier, Rudolf K. Thauer, Julia A. Vorholt, Meike Goenrich, Ulrike Demmer, Ulrich ErmlerAbstract:Tetrahydromethanopterin (H4 MPT) is a tetrahydrofolate analogue involved as a C1 carrier in the metabolism of various groups of microorganisms. How H4MPT is bound to the respective C1 unit converting enzymes remained elusive. We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 angstrom without and at 1.9 angstrom with methylene-H4MPT bound. Methylene-H4MPT is bound in an "S"-shaped conformation into the cleft formed between two adjacent subunits. Coenzyme binding is accompanied by side chain rearrangements up to 5 angstrom and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster, and an inversion of the amide side chain of Gln88. Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70 degrees that is more pronounced than that reported for free methylene-H4MPT in solution (50 degrees). Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyzes the formation of methylene-H4MPT from H4MPT and formaldehyde. The molecular mechanism ofthis reaction involving His22 as acid catalyst is discussed.
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Tetrahydromethanopterin specific enzymes from methanopyrus kandleri
Methods in Enzymology, 2001Co-Authors: Seigo Shima, Rudolf K. ThauerAbstract:Publisher Summary Methanopyrus kandleri is a hyperthermophilic archaeon growing optimally at 98° on H 2 and CO 2 with the formation of CH 4 . The organism belonging to the kingdom of Euryarchaeota is the most thermophilic methanogen known so far and is phylogenetically only distantly related to all other known methanogens. The pathway of CO 2 reduction to CH 4 in M. kandleri has been shown to be identical to that used in all other methanogens. It involves six Tetrahydromethanopterin-specific enzymes. Tetrahydromethanopterin (H 4 MPT) is a tetrahydrofolate analog. This chapter describes the purification, assay, and properties of the five characterized H 4 MPT-specific enzymes from M. kandleri . It also provides a description of the isolation of the coenzymes required to assay these enzymes.
Ulrich Ermler - One of the best experts on this subject based on the ideXlab platform.
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the bacterial fe hydrogenase paralog uses tetrahydrofolate derivatives as substrates
Angewandte Chemie, 2019Co-Authors: Tomohiro Watanabe, Gangfeng Huang, Kenichi Ataka, Ulrich Ermler, T Wagner, Jörg Kahnt, Seigo ShimaAbstract:: [Fe]-hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl-Tetrahydromethanopterin (methenyl-H4 MPT+ ) with H2 . H4 MPT is a C1-carrier of methanogenic archaea. One bacterial genus, Desulfurobacterium, contains putative genes for the Hmd paralog, termed HmdII, and the HcgA-G proteins. The latter are required for the biosynthesis of the prosthetic group of Hmd, the iron-guanylylpyridinol (FeGP) cofactor. This finding is intriguing because Hmd and HmdII strictly use H4 MPT derivatives that are absent in most bacteria. We identified the presence of the FeGP cofactor in D. thermolithotrophum. The bacterial HmdII reconstituted with the FeGP cofactor catalyzed the hydrogenation of derivatives of tetrahydrofolate, the bacterial C1-carrier, albeit with low enzymatic activities. The crystal structures show how Hmd recognizes tetrahydrofolate derivatives. These findings have an impact on future biotechnology by identifying a bacterial Hmd paralog.
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Molecular characterization of methanogenic N(5)-methyl-Tetrahydromethanopterin: Coenzyme M methyltransferase.
Biochimica et biophysica acta, 2016Co-Authors: Vikrant Upadhyay, Seigo Shima, Katharina Ceh, Franz Tumulka, Rupert Abele, Jan Hoffmann, Julian D. Langer, Ulrich ErmlerAbstract:Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-Tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-Tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430 kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.
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structure and catalytic mechanism of n 5 n 10 methenyl Tetrahydromethanopterin cyclohydrolase
Biochemistry, 2012Co-Authors: Vikrant Upadhyay, Seigo Shima, Ulrike Demmer, Eberhard Warkentin, Johanna Moll, Ulrich ErmlerAbstract:MethenylTetrahydromethanopterin (methenyl-H4MPT+) cyclohydrolase (Mch) catalyzes the interconversion of methenyl-H4MPT+ and formyl-H4MPT in the one-carbon energy metabolism of methanogenic, methano...
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the structure of formylmethanofuran Tetrahydromethanopterin formyltransferase in complex with its coenzymes
Journal of Molecular Biology, 2006Co-Authors: Priyamvada Acharya, Rudolf K. Thauer, Ulrich Ermler, Eberhard Warkentin, Seigo ShimaAbstract:Formylmethanofuran:Tetrahydromethanopterin formyltransferase is an essential enzyme in the one-carbon metabolism of methanogenic and sulfate-reducing archaea and of methylotrophic bacteria. The enzyme, which is devoid of a prosthetic group, catalyzes the reversible formyl transfer between the two substrates coenzyme methanofuran and coenzyme Tetrahydromethanopterin (H 4 MPT) in a ternary complex catalytic mechanism. The structure of the formyltransferase without its coenzymes has been determined earlier. We report here the structure of the enzyme in complex with both coenzymes at a resolution of 2.0 A. Methanofuran, characterized for the first time in an enzyme structure, is embedded in an elongated cleft at the homodimer interface and fixed by multiple hydrophobic interactions. In contrast, Tetrahydromethanopterin is only weakly bound in a shallow and wide cleft that provides two binding sites. It is assumed that the binding of the bulky coenzymes induces conformational changes of the polypeptide in the range of 3 A that close the H 4 MPT binding cleft and position the reactive groups of both substrates optimally for the reaction. The key residue for substrate binding and catalysis is the strictly conserved Glu245. Glu245, embedded in a hydrophobic region and completely buried upon Tetrahydromethanopterin binding, is presumably protonated prior to the reaction and is thus able to stabilize the tetrahedral oxyanion intermediate generated by the nucleophilic attack of the N 5 atom of Tetrahydromethanopterin onto the formyl carbon atom of formylmethanofuran.
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how an enzyme binds the c1 carrier Tetrahydromethanopterin structure of the Tetrahydromethanopterin dependent formaldehyde activating enzyme fae from methylobacterium extorquens am1
Journal of Biological Chemistry, 2005Co-Authors: Priyamvada Acharya, Christoph H. Hagemeier, Rudolf K. Thauer, Julia A. Vorholt, Meike Goenrich, Ulrike Demmer, Ulrich ErmlerAbstract:Tetrahydromethanopterin (H4 MPT) is a tetrahydrofolate analogue involved as a C1 carrier in the metabolism of various groups of microorganisms. How H4MPT is bound to the respective C1 unit converting enzymes remained elusive. We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 angstrom without and at 1.9 angstrom with methylene-H4MPT bound. Methylene-H4MPT is bound in an "S"-shaped conformation into the cleft formed between two adjacent subunits. Coenzyme binding is accompanied by side chain rearrangements up to 5 angstrom and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster, and an inversion of the amide side chain of Gln88. Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70 degrees that is more pronounced than that reported for free methylene-H4MPT in solution (50 degrees). Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyzes the formation of methylene-H4MPT from H4MPT and formaldehyde. The molecular mechanism ofthis reaction involving His22 as acid catalyst is discussed.
Julia A. Vorholt - One of the best experts on this subject based on the ideXlab platform.
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how an enzyme binds the c1 carrier Tetrahydromethanopterin structure of the Tetrahydromethanopterin dependent formaldehyde activating enzyme fae from methylobacterium extorquens am1
Journal of Biological Chemistry, 2005Co-Authors: Priyamvada Acharya, Christoph H. Hagemeier, Rudolf K. Thauer, Julia A. Vorholt, Meike Goenrich, Ulrike Demmer, Ulrich ErmlerAbstract:Tetrahydromethanopterin (H4 MPT) is a tetrahydrofolate analogue involved as a C1 carrier in the metabolism of various groups of microorganisms. How H4MPT is bound to the respective C1 unit converting enzymes remained elusive. We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 angstrom without and at 1.9 angstrom with methylene-H4MPT bound. Methylene-H4MPT is bound in an "S"-shaped conformation into the cleft formed between two adjacent subunits. Coenzyme binding is accompanied by side chain rearrangements up to 5 angstrom and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster, and an inversion of the amide side chain of Gln88. Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70 degrees that is more pronounced than that reported for free methylene-H4MPT in solution (50 degrees). Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyzes the formation of methylene-H4MPT from H4MPT and formaldehyde. The molecular mechanism ofthis reaction involving His22 as acid catalyst is discussed.
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multiple formate dehydrogenase enzymes in the facultative methylotroph methylobacterium extorquens am1 are dispensable for growth on methanol
Journal of Bacteriology, 2004Co-Authors: Ludmila Chistoserdova, Julia A. Vorholt, Markus Laukel, Jeancharles Portais, Mary E. LidstromAbstract:The classic scheme of energy metabolism during methylotrophic growth involves a formate oxidation step except in strains in which all formaldehyde is oxidized in the cyclic ribulose monophosphate cycle (1). Formate dehydrogenase (FDH) activity has been detected in most methylotrophs (3, 10, 13, 15, 22, 42), and a few FDHs have been purified and analyzed (reviewed in reference 39). In the methylotrophic yeast Candida boidinii, the FDH step was shown not to be essential for methylotrophic growth, but FDH mutants showed reduced growth on methanol (32). However, as the complete C. boidinii genome sequence is not available, the presence of other FDHs is not excluded. Mutant-based analysis of the role of the FDH step in C1 oxidation has not yet been attempted in methylotrophic bacteria. M. extorquens AM1 offers a convenient model to study this question. It possesses two pathways in which formaldehyde can be oxidized to formate (Fig. (Fig.1),1), one linked to Tetrahydromethanopterin (H4MPT) and another linked to tetrahydrofolate (H4F) (5, 6). The enzymes involved in the two pathways have been studied in detail, and current evidence suggests that the main pathway for oxidizing formaldehyde is the H4MPT-linked pathway (reviewed in reference 37). It has been demonstrated recently that this pathway produces formate as an intermediate, a result of a formylmethanofuran transferase/hydrolase reaction (29), and thus in this pathway one molecule of formate is formed in M. extorquens AM1 per oxidized molecule of a C1 substrate, such as methanol or methylamine. This formate is subsequently oxidized to CO2, presumably by FDH (Fig. (Fig.11). FIG. 1. C1 metabolism of M. extorquens AM1. H4MPT, Tetrahydromethanopterin; H4F, tetrahydrofolate; Fae, H4MPT-dependent formaldehyde activating enzyme (39); MtdA, NADP-dependent methylene-H4MPT dehydrogenase (8, 38); MtdB, NAD(P)-dependent methylene-H4MPT dehydrogenase ... An FDH from M. extorquens AM1 has recently been purified and characterized and shown to be a novel, tungsten-containing FDH encoded by two genes, fdh1AB (20). In this study we identified two new regions in the M. extorquens AM1 chromosome coding for two additional FDH enzymes. Using mutation analysis, we demonstrate that all three enzymes are expressed during growth on C1 compounds but none is essential for growth on methanol, providing new insight into the energetics of C1 metabolism in serine cycle methylotrophs.
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generation of formate by the formyltransferase hydrolase complex fhc from methylobacterium extorquens am1
FEBS Letters, 2002Co-Authors: Olivier Saurel, Barbara K Pomper, Alain Milon, Julia A. VorholtAbstract:Methylobacterium extorquens AM1 possesses a formyltransferase (Ftr) complex that is essential for growth in the presence of methanol and involved in formaldehyde oxidation to CO2. One of the subunits of the complex carries the catalytic site for transfer of the formyl group from Tetrahydromethanopterin to methanofuran (MFR). We now found via nuclear magnetic resonance-based studies that the Ftr complex also catalyzes the hydrolysis of formyl-MFR and generates formate. The enzyme was therefore renamed Ftr/hydrolase complex (Fhc). FhcA shares a sequence pattern with amidohydrolases and is assumed to be the catalytic site where the hydrolysis takes place.
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Purification and characterization of the methylene Tetrahydromethanopterin dehydrogenase MtdB and the methylene tetrahydrofolate dehydrogenase FolD from Hyphomicrobium zavarzinii ZV580
Archives of microbiology, 2002Co-Authors: Meike Goenrich, Jan Bursy, Eva Hübner, Dietmar Linder, Arnold C. Schwartz, Julia A. VorholtAbstract:Recently, it has been shown that heterotrophic methylotrophic Proteobacteria contain tetrahydrofolate (H4F)- and Tetrahydromethanopterin (H4MPT)-dependent enzymes. Here we report on the purification of two methylene tetrahydropterin dehydrogenases from the methylotroph Hyphomicrobium zavarzinii ZV580. Both dehydrogenases are composed of one type of subunit of 31 kDa. One of the dehydrogenases is NAD(P)-dependent and specific for methylene H4MPT (specific activity: 680 U/mg). Its N-terminal amino acid sequence showed sequence identity to NAD(P)-dependent methylene H4MPT dehydrogenase MtdB from Methylobacterium extorquens AM1. The second dehydrogenase is specific for NADP and methylene H4F (specific activity: 180 U/mg) and also exhibits methenyl H4F cyclohydrolase activity. Via N-terminal amino acid sequencing this dehydrogenase was identified as belonging to the classical bifunctional methylene H4F dehydrogenases/cyclohydrolases (FolD) found in many bacteria and eukarya. Apparently, the occurrence of methylene tetrahydrofolate and methylene Tetrahydromethanopterin dehydrogenases is not uniform among different methylotrophic α-Proteobacteria. For example, FolD was not found in M. extorquens AM1, and the NADP-dependent methylene H4MPT dehydrogenase MtdA was present in the bacterium that also shows H4F activity.
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Characterization of the formyltransferase from Methylobacterium extorquens AM1
European journal of biochemistry, 2001Co-Authors: Barbara K Pomper, Julia A. VorholtAbstract:1 Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylmethanofuran–Tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and Tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 µm; apparent Km for H4MPT = 30 µm). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of ≈ 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a [4Fe−4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.
Ludmila Chistoserdova - One of the best experts on this subject based on the ideXlab platform.
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the distribution and evolution of c1 transfer enzymes and evolution of the planctomycetes
2013Co-Authors: Ludmila ChistoserdovaAbstract:When the first genome sequence of a Planctomycete became available (in 2003), it revealed the presence of genes encoding a pathway for a Tetrahydromethanopterin-mediated transfer of C1 units between the oxidation levels of formaldehyde and formate, resembling a pathway for methanogenesis being carried out by a specialized group of Archaea and a pathway for formaldehyde oxidation employed by some methylotrophic bacteria, the latter pathway acting in reverse to methanogenesis. This discovery was of importance as the presence of the genes in question in the Planctomycetes has challenged the assumption of a limited distribution of these genes/pathways in the microbial world, at the same time suggesting novel scenarios for the evolution of C1 transfer pathways in microbes and providing support for the potential antiquity of these pathways. In this chapter, I review the early work on the discovery and analysis of the genetic determinants of C1 functions in Planctomycetes and the significance of these discoveries in interpreting the emergence and the evolution of C1 metabolism in Prokaryotes. This is followed by a review of the continuously emerging new genomic data suggesting a much wider distribution of the Tetrahydromethanopterin-linked functions in Prokaryotes, further supporting the hypothesis of the long evolution for these functions. While the Planctomycetes provide these important insights into the evolution of specific biochemical pathways as well as the evolution of Prokaryotes in general, the exact function of the Tetrahydromethanopterin-linked C1 transfer pathway in Planctomycetes and in many other phyla remains unknown.
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MtdC, a Novel Class of Methylene Tetrahydromethanopterin Dehydrogenases
Journal of Bacteriology, 2005Co-Authors: Marina G. Kalyuzhnaya, Christoph H. Hagemeier, Ludmila ChistoserdovaAbstract:Novel methylene Tetrahydromethanopterin (H4MPT) dehydrogenase enzymes, named MtdC, were purified after expressing in Escherichia coli genes from, respectively, Gemmata sp. strain Wa1-1 and environmental DNA originating from unidentified microbial species. The MtdC enzymes were shown to possess high affinities for methylene-H4MPT and NADP but low affinities for methylene tetrahydrofolate or NAD. The substrate range and the kinetic properties revealed by MtdC enzymes distinguish them from the previously characterized bacterial methylene-H4MPT dehydrogenases, MtdA and MtdB. While revealing higher sequence similarity to MtdA enzymes, MtdC enzymes appear to fulfill a function homologous to the function of MtdB, as part of the H4MPT-linked pathway for formaldehyde oxidation/detoxification.
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Analysis of fae and fhcD Genes in Mono Lake, California
2005Co-Authors: Olivier Nercessian, Marina G. Kalyuzhnaya, Mary E. Lidstrom, Samantha B. Joye, Ludmila ChistoserdovaAbstract:Genes for two enzymes of the Tetrahydromethanopterin-linked C1 transfer pathway (fae and fhcD) were detected in hypersaline, hyperalkalineMono Lake (California), via PCR amplification and analysis. Low diversity for fae and fhcDwas noted, in contrast to the diversity previously detected in a freshwater lake, LakeWashington (Washington). Methylotrophic bacteria are a group of organisms that con-sume a wide range of C1 compounds, such as methane, meth-anol, methylated amines, methylated glycines, halomethanes, and methylated sulfur species (1, 17). They are found in a variety of environments, such as freshwater, marine, and ter-restrial habitats, as well as habitats characterized by extreme conditions, such as highly saline, alkaline, or acidic habitats (2
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Multiple formaldehyde oxidation/detoxification pathways in Burkholderia fungorum LB400
2004Co-Authors: Christopher J. Marx, Ludmila Chistoserdova, Jonathan A. Miller, Mary E. LidstromAbstract:Burkholderia species are free-living bacteria with a versatile metabolic lifestyle. The genome of B. fungorum LB400 is predicted to encode three different pathways for formaldehyde oxidation: an NAD-linked, glutathione (GSH)-independent formaldehyde dehydrogenase; an NAD-linked, GSH-dependent formaldehyde oxidation system; and a Tetrahydromethanopterin-methanofuran-dependent formaldehyde oxidation system. The other Burkholderia species for which genome sequences are available, B. mallei, B. pseudomallei, and B. cepacia, are predicted to contain only the first two of these pathways. The roles of the three putative formaldehyde oxidation pathways in B. fungorum LB400 have been assessed via knockout mutations in each of these pathways, as well as in all combinations of knockouts. The resulting mutants have the expected loss of enzyme activities and exhibit defects of varying degrees of severity during growth on choline, a formaldehyde-producing substrate. Our data suggest that all three pathways are involved in formaldehyde detoxification and are functionally redundant under the tested conditions. The lifestyle of free-living organisms involves many challenges related to both seasonal and sudden changes in nutrient supply, temperature, salinity, etc., and in some cases it appears that a correlation exists between the versatility of the lifestyl
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multiple formate dehydrogenase enzymes in the facultative methylotroph methylobacterium extorquens am1 are dispensable for growth on methanol
Journal of Bacteriology, 2004Co-Authors: Ludmila Chistoserdova, Julia A. Vorholt, Markus Laukel, Jeancharles Portais, Mary E. LidstromAbstract:The classic scheme of energy metabolism during methylotrophic growth involves a formate oxidation step except in strains in which all formaldehyde is oxidized in the cyclic ribulose monophosphate cycle (1). Formate dehydrogenase (FDH) activity has been detected in most methylotrophs (3, 10, 13, 15, 22, 42), and a few FDHs have been purified and analyzed (reviewed in reference 39). In the methylotrophic yeast Candida boidinii, the FDH step was shown not to be essential for methylotrophic growth, but FDH mutants showed reduced growth on methanol (32). However, as the complete C. boidinii genome sequence is not available, the presence of other FDHs is not excluded. Mutant-based analysis of the role of the FDH step in C1 oxidation has not yet been attempted in methylotrophic bacteria. M. extorquens AM1 offers a convenient model to study this question. It possesses two pathways in which formaldehyde can be oxidized to formate (Fig. (Fig.1),1), one linked to Tetrahydromethanopterin (H4MPT) and another linked to tetrahydrofolate (H4F) (5, 6). The enzymes involved in the two pathways have been studied in detail, and current evidence suggests that the main pathway for oxidizing formaldehyde is the H4MPT-linked pathway (reviewed in reference 37). It has been demonstrated recently that this pathway produces formate as an intermediate, a result of a formylmethanofuran transferase/hydrolase reaction (29), and thus in this pathway one molecule of formate is formed in M. extorquens AM1 per oxidized molecule of a C1 substrate, such as methanol or methylamine. This formate is subsequently oxidized to CO2, presumably by FDH (Fig. (Fig.11). FIG. 1. C1 metabolism of M. extorquens AM1. H4MPT, Tetrahydromethanopterin; H4F, tetrahydrofolate; Fae, H4MPT-dependent formaldehyde activating enzyme (39); MtdA, NADP-dependent methylene-H4MPT dehydrogenase (8, 38); MtdB, NAD(P)-dependent methylene-H4MPT dehydrogenase ... An FDH from M. extorquens AM1 has recently been purified and characterized and shown to be a novel, tungsten-containing FDH encoded by two genes, fdh1AB (20). In this study we identified two new regions in the M. extorquens AM1 chromosome coding for two additional FDH enzymes. Using mutation analysis, we demonstrate that all three enzymes are expressed during growth on C1 compounds but none is essential for growth on methanol, providing new insight into the energetics of C1 metabolism in serine cycle methylotrophs.
Mary E. Lidstrom - One of the best experts on this subject based on the ideXlab platform.
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elucidation of the role of the methylene Tetrahydromethanopterin dehydrogenase mtda in the Tetrahydromethanopterin dependent oxidation pathway in methylobacterium extorquens am1
Journal of Bacteriology, 2013Co-Authors: N C Martinezgomez, S Nguyen, Mary E. LidstromAbstract:The methylotroph Methylobacterium extorquens AM1 oxidizes methanol and methylamine to formaldehyde and subsequently to formate, an intermediate that serves as the branch point between assimilation (formation of biomass) and dissimilation (oxidation to CO₂). The oxidation of formaldehyde to formate is dephosphoTetrahydromethanopterin (dH₄MPT) dependent, while the assimilation of carbon into biomass is tetrahydrofolate (H₄F) dependent. This bacterium contains two different enzymes, MtdA and MtdB, both of which are dehydrogenases able to use methylene-dH₄MPT, an intermediate in the oxidation of formaldehyde to formate. Unique to MtdA is a second enzymatic activity with methylene-H₄F. Since methylene-H₄F is the entry point into the biomass pathways, MtdA plays a key role in assimilatory metabolism. However, its role in oxidative metabolism via the dH₄MPT-dependent pathway and its apparent inability to replace MtdB in vivo on methanol growth are not understood. Here, we have shown that an mtdB mutant is able to grow on methylamine, providing a system to study the role of MtdA. We demonstrate that the absence of MtdB results in the accumulation of methenyl-dH₄MPT. Methenyl-dH₄MPT is shown to be a competitive inhibitor of the reduction of methenyl-H₄F to methylene-H₄F catalyzed by MtdA, with an estimated Ki of 10 μM. Thus, methenyl-dH₄MPT accumulation inhibits H₄F-dependent assimilation. Overexpression of mch in the mtdB mutant strain, predicted to reduce methenyl-dH₄MPT accumulation, enhances growth on methylamine. Our model proposes that MtdA regulates carbon flux due to differences in its kinetic properties for methylene-dH₄MPT and for methenyl-H₄F during growth on single-carbon compounds.
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Formaldehyde Metabolism of M. extorquens AM1
2013Co-Authors: Christopher J. Marx, Stephen J Van Dien, Mary E. LidstromAbstract:Three modules work to provide two cellular outputs: formaldehyde assimilation and dissimilation. The direct condensation of formaldehyde with H4F is shown in green. A second proposed route for generating methylene-tetrahydrofolate (methylene-H4F), the consecutive action of the H4MPT and H4F modules is shown in blue. Fae, formaldehyde activating enzyme; Fch, methenyl H4F cyclohydrolase; FDH, formate dehydrogenase; Fhc, formyltransferase/hydrolase complex; FtfL, formyl H4F ligase; H4MPT, Tetrahydromethanopterin; Mch, methenyl H4MPT cyclohydrolase; MDH, methanol dehydrogenase; MtdA, methylene H4F/H4MPT dehydrogenase; MtdB, methylene H4MPT dehydrogenase. Spontaneous and reversible reactions are indicated.
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Analysis of fae and fhcD Genes in Mono Lake, California
2005Co-Authors: Olivier Nercessian, Marina G. Kalyuzhnaya, Mary E. Lidstrom, Samantha B. Joye, Ludmila ChistoserdovaAbstract:Genes for two enzymes of the Tetrahydromethanopterin-linked C1 transfer pathway (fae and fhcD) were detected in hypersaline, hyperalkalineMono Lake (California), via PCR amplification and analysis. Low diversity for fae and fhcDwas noted, in contrast to the diversity previously detected in a freshwater lake, LakeWashington (Washington). Methylotrophic bacteria are a group of organisms that con-sume a wide range of C1 compounds, such as methane, meth-anol, methylated amines, methylated glycines, halomethanes, and methylated sulfur species (1, 17). They are found in a variety of environments, such as freshwater, marine, and ter-restrial habitats, as well as habitats characterized by extreme conditions, such as highly saline, alkaline, or acidic habitats (2
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Multiple formaldehyde oxidation/detoxification pathways in Burkholderia fungorum LB400
2004Co-Authors: Christopher J. Marx, Ludmila Chistoserdova, Jonathan A. Miller, Mary E. LidstromAbstract:Burkholderia species are free-living bacteria with a versatile metabolic lifestyle. The genome of B. fungorum LB400 is predicted to encode three different pathways for formaldehyde oxidation: an NAD-linked, glutathione (GSH)-independent formaldehyde dehydrogenase; an NAD-linked, GSH-dependent formaldehyde oxidation system; and a Tetrahydromethanopterin-methanofuran-dependent formaldehyde oxidation system. The other Burkholderia species for which genome sequences are available, B. mallei, B. pseudomallei, and B. cepacia, are predicted to contain only the first two of these pathways. The roles of the three putative formaldehyde oxidation pathways in B. fungorum LB400 have been assessed via knockout mutations in each of these pathways, as well as in all combinations of knockouts. The resulting mutants have the expected loss of enzyme activities and exhibit defects of varying degrees of severity during growth on choline, a formaldehyde-producing substrate. Our data suggest that all three pathways are involved in formaldehyde detoxification and are functionally redundant under the tested conditions. The lifestyle of free-living organisms involves many challenges related to both seasonal and sudden changes in nutrient supply, temperature, salinity, etc., and in some cases it appears that a correlation exists between the versatility of the lifestyl
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multiple formate dehydrogenase enzymes in the facultative methylotroph methylobacterium extorquens am1 are dispensable for growth on methanol
Journal of Bacteriology, 2004Co-Authors: Ludmila Chistoserdova, Julia A. Vorholt, Markus Laukel, Jeancharles Portais, Mary E. LidstromAbstract:The classic scheme of energy metabolism during methylotrophic growth involves a formate oxidation step except in strains in which all formaldehyde is oxidized in the cyclic ribulose monophosphate cycle (1). Formate dehydrogenase (FDH) activity has been detected in most methylotrophs (3, 10, 13, 15, 22, 42), and a few FDHs have been purified and analyzed (reviewed in reference 39). In the methylotrophic yeast Candida boidinii, the FDH step was shown not to be essential for methylotrophic growth, but FDH mutants showed reduced growth on methanol (32). However, as the complete C. boidinii genome sequence is not available, the presence of other FDHs is not excluded. Mutant-based analysis of the role of the FDH step in C1 oxidation has not yet been attempted in methylotrophic bacteria. M. extorquens AM1 offers a convenient model to study this question. It possesses two pathways in which formaldehyde can be oxidized to formate (Fig. (Fig.1),1), one linked to Tetrahydromethanopterin (H4MPT) and another linked to tetrahydrofolate (H4F) (5, 6). The enzymes involved in the two pathways have been studied in detail, and current evidence suggests that the main pathway for oxidizing formaldehyde is the H4MPT-linked pathway (reviewed in reference 37). It has been demonstrated recently that this pathway produces formate as an intermediate, a result of a formylmethanofuran transferase/hydrolase reaction (29), and thus in this pathway one molecule of formate is formed in M. extorquens AM1 per oxidized molecule of a C1 substrate, such as methanol or methylamine. This formate is subsequently oxidized to CO2, presumably by FDH (Fig. (Fig.11). FIG. 1. C1 metabolism of M. extorquens AM1. H4MPT, Tetrahydromethanopterin; H4F, tetrahydrofolate; Fae, H4MPT-dependent formaldehyde activating enzyme (39); MtdA, NADP-dependent methylene-H4MPT dehydrogenase (8, 38); MtdB, NAD(P)-dependent methylene-H4MPT dehydrogenase ... An FDH from M. extorquens AM1 has recently been purified and characterized and shown to be a novel, tungsten-containing FDH encoded by two genes, fdh1AB (20). In this study we identified two new regions in the M. extorquens AM1 chromosome coding for two additional FDH enzymes. Using mutation analysis, we demonstrate that all three enzymes are expressed during growth on C1 compounds but none is essential for growth on methanol, providing new insight into the energetics of C1 metabolism in serine cycle methylotrophs.
