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N 5-Methyltetrahydromethanopterin: coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein

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Abstract

An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N 5-methyltetrahydromethanopterin (CH3−H4MPT) to coenzyme M (H−S−CoM) in methanogenic archaebacteria. With this method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri and in H2/CO2-grown Methanobacterium thermoautotrophicum were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH3−S−CoM formation from CH3−H4MPT (apparent K m=50 μM) and H−S−CoM (apparent K m=250 μM) of approximately 0.6 μmol·min-1·mg protein-1. For activity the presence of Ti(III) citrate (apparent K m=15 μM) and of ATP (apparent K m=30 μM) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH3−H4MPT in the absence of H−S−CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH3−H4MPT to H−S−CoM in the absence of ATP and Ti(III). Demethylation in the presence of H−S−CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.

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Abbreviations

H4MPT:

tetrahydromethanopterin

CH3−H4MPT:

N 5-methyl-H4MPT

H−S−CoM:

2-mercaptoethanesulfonate or coenzyme M

CH3−S−CoM:

2(methylthio)ethanesulfonate or methylcoenzyme M

SDS-PAGE:

sodium dodecylsulfate polyacrylamide gel electrophoresis

DTT:

dithiothreitol

MOPS:

morpholinopropanesulfonate

CHAPS:

3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate

1 U =:

1 μmol/min

References

  • BanerjeeRV, FrascaV, BallouDP, MatthewsRG (1990a) Participation of cob(I) alamin in the reaction catalyzed by methionine synthase from Escherichia coli: a steady-state and rapid reaction kinetic analysis. Biochemistry 29: 11 101–11 109

    Google Scholar 

  • BanerjeeRV, HarderSR, RagsdaleSW, MatthewsRG (1990b) Mechanism of reductive activation of cobalamin-dependent methionine synthase: An electron paramagnetic resonance spectroelectrochemical study. Biochemistry 29: 1129–1135

    Google Scholar 

  • BaronSF, FerryJG (1989) Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum. J Bacteriol 171: 3846–3853

    Google Scholar 

  • BobikTA, WolfeRS (1989) Activation of formylmethanofuran synthesis in cell extracts of Methanobacterium thermoautotrophicum. J Bacteriol 171: 1423–1427

    Google Scholar 

  • Börner G (1988) Isolierung von vier Coenzymen der Methanogenese aus Methanobacterium thermoautotrophicum. Diploma thesis, Department of Biology, University of Marburg

  • BottM, ThauerRK (1987) Proton-motive-force-driven formation of CO from CO2 and H2 in meantanogenic bacteria. Eur J Biochem 168: 407–412

    Google Scholar 

  • BradfordMM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254

    Google Scholar 

  • BrömmelstroetBWte, HensgensCMH, KeltjensJT, DriftCvan der, VogelsGD (1990) Purification and properties of 5,10-methylenetetrahydromethanopterin reductase, a coenzyme F420-dependent enzyme, from Methanobacterium thermoautotrophicum strain ΔH. J Biol Chem 265: 1852–1856

    Google Scholar 

  • CaoX, KrzyckiJA (1991) Acetate-dependent methylation of two corrinoid proteins in extracts of Methanosarcina barkeri. J Bacteriol 173: 5439–5448

    Google Scholar 

  • DangelW, SchulzH, DiekertG, KönigH, FuchsG (1987) Occurrence of corrinoid-containing membrane proteins in anaerobic bacteria. Arch Microbiol 148: 52–56

    Google Scholar 

  • DeppenmeierU, BlautM, MahlmannA, GottschalkG (1990) Reduced coenzyme F420: heterodisulfide oxidoreductase, a proton-translocating redox system in methanogenic bacteria. Proc Natl Acad Sci USA 87: 9449–9453

    Google Scholar 

  • DeppenmeierU, BlautM, GottschalkG (1991) H2: heterodisulfide oxidoreductase, second energy-conserving system in the methanogenic strain Gö1. Arch Microbiol 155: 272–277

    Google Scholar 

  • DiMarcoAA, BobikTA, WolfeRS (1990) Unusual coenzymes of methanogenesis. Annu Rev Biochem 59: 355–394

    Google Scholar 

  • Escalante-SemerenaJC, RinehartKL, JrWolfeRS (1984) Tetrahydromethanopterin, a carbon carrier in methanogenesis. J Biol Chem 259: 9447–9455

    Google Scholar 

  • EvansHJ, HarkerAR, PapenH, RussellSA, HanusFJ, ZuberM (1987) Physiology, biochemistry, and genetics of the uptake hydrogenase in Rhizobia. Ann Rev Microbiol 41: 335–361

    Google Scholar 

  • FiebigK, FriedrichB, (1989) Purification of the F420-reducing hydrogenase from Methanosarcina barkeri (strain Fusaro). Eur J Biochem 184: 79–88

    Google Scholar 

  • FischerR, ThauerRK (1989) Methyltetrahydromethanopterin as an intermediate in methanogenesis from acetate in Methanosarcina barkeri. Arch Microbiol 151: 459–465

    Google Scholar 

  • FischerR, ThauerRK (1990) Methanogenesis from acetate in cell extracts of Methanosarcina barkeri: isotope exchange between CO2 and the carbonyl group of acetyl-CoA, and the role of H2. Arch Microbiol 153: 156–162

    Google Scholar 

  • FoxJA, LivingstonDJ, Orme-JohnsonWH, WalshCT (1987) 8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 1. Purification and characterization. Biochemistry 26: 4219–4227

    Google Scholar 

  • FrascaV, BanerjeeRV, DunhamWR, SandsRH, MatthewsRG (1988) Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme. Biochemistry 27: 8458–8465

    Google Scholar 

  • GrahameDA (1989) Different isozymes of methylcobalamin: 2-mercaptoethanesulfonate methyltransferase predominate in methanol-versus actate-grown Methanosarcina barkeri. J Biol Chem 264: 12890–12894

    Google Scholar 

  • HarderSR, LuWP, FeinbergBA, RagsdaleSW (1989) Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum. Biochemistry 28: 9080–9087

    Google Scholar 

  • HedderichR, BerkesselA, ThauerRK (1989) Catalytic properties of the heterodisulfide reductase involved in the final step of methanogenesis. FEBS Lett 255: 67–71

    Google Scholar 

  • HedderichR, BerkesselA, ThauerRK (1990) Purification and properties of heterodisulfide reductase from Methanobacterium thermoautotrophicum (strain Marburg). Eur J Biochem 193: 255–261

    Google Scholar 

  • InatomiKI, MaedaM, FutaiM (1989) Dicyclohexylcarbodiimide-binding protein is a subunit of the Methanosarcina barkeri ATPase complex. Biochem Biophys Res Commun 162: 1585–1590

    Google Scholar 

  • KaeslerB, SchönheitP (1989) The sodium cycle in methanogenesis. CO2 reduction to the formaldehyde level in methanogenic bacteria is driven by a primary electrochemical potential of Na+ generated by formaldehyde reduction to CH4. Eur J Biochem 186: 309–316

    Google Scholar 

  • KarraschM, BottM, ThauerRK (1989) Carbonic anhydrase activity in acetate grown Methanosarcina barkeri. Arch Microbiol 151: 137–142

    Google Scholar 

  • KeltjensJT, CaertelingGC, VogelsGD (1986) Methanopterin and tetrahydromethanopterin derivatives: isolation, synthesis, and identification by high-performance liquid chromatography. In: ColoweckKaplan (eds) Methods of enzymology, vol 122. Academic Press, Orlando, pp 412–425

    Google Scholar 

  • KeltjensJT, BrömmelstroetBWte, KengenSWM, DriftCvan der, VogelsGD (1990) 5,6,7,8-Tetrahydromethanopterin-dependent enzymes involved in methanogenesis. FEMS Microbiol Rev 87: 327–332

    Google Scholar 

  • KengenSWM, MosterdJJ, NelissenRLH, KeltjensJT, DriftCvan der, VogelsGD (1988) Reductive activation of the methyltetrahydromethanopterin: coenzyme M methyltransferase from Methanobacterium thermoautotrophicum strain ΔH. Arch Microbiol 150: 405–412

    Google Scholar 

  • KengenSWM, DaasPJH, KeltjensJT, DriftCvan der, VogelsGD (1990) Stimulation of the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in cell-free extracts of Methanobacterium thermoautotrophicum by the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate. Arch Microbiol 154: 156–161

    Google Scholar 

  • KroneUE, ThauerRK, HogenkampHPC (1989) Reductive dehalogenation of chlorinated C1-hydrocarbons mediated by corrinoids. Biochemistry 28: 4908–4914

    Google Scholar 

  • KühnW, GottschalkG (1983) Characterization of the cytochromes occuring in Methanosarcina species. Eur J Biochem 135: 89–94

    Google Scholar 

  • MaK, ThauerRK (1990a) Purification and properties of N 5,N 10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg). Eur J Biochem 1991: 187–193

    Google Scholar 

  • MaK, ThauerRK (1990b) N 5,N 10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri. FEMS Microbiol Lett 70: 119–124

    Google Scholar 

  • MaK, LinderD, StetterKO, ThauerRK (1991) Purification and properties of N 5,N 10-methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) from the extreme thermophile Methanopyrus kandleri. Arch Microbiol 155: 593–600

    Google Scholar 

  • MeijdenPvan der, HeythuysenHJ, PouwelsA, HouwenF, DriftCvan der, VogelsGD (1983) Methyltransferases involved in methanol conversion by Methanosarcina barkeri. Arch Microbiol 134: 238–242

    Google Scholar 

  • MeijdenPvan der, BrömmelstroetBWte, PoirotCM, DriftCvan der, VogelsGD (1984a) Purification and properties of methanol: 5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarcina barkeri. J Bacteriol 160: 629–635

    Google Scholar 

  • MeijdenPvan der, DriftCvan der, VogelsGD (1984b) Methanol conversion in Eubacterium limosum. Arch Microbiol 138: 360–364

    Google Scholar 

  • MüllerV, BlautM, HeiseR, WinnerC, GottschalkG (1990) Sodium bioenergetics in methanogens and acetogens. FEMS Microbiol Rev 87: 373–376

    Google Scholar 

  • MüllerV, WinnerC, GottschalkG (1988) Electron-transport-driven sodium extrusion during methanogenesis from formaldehyde and molecular hydrogen by Methanosarcina barkeri. Eur J Biochem 178: 519–525

    Google Scholar 

  • MuthE, MörschelE, KleinA (1987) Purification and characterization of an 8-hydroxy-5-deazaflavin-reducing hydrogenase from the archaebacterium Methanococcus voltae. Eur J Biochem 169: 571–577

    Google Scholar 

  • PeinemannS, HedderichR, BlautM, ThauerRK, GottschalkG (1990) ATP synthesis coupled to electron transfer from H2 to the heterodisulfide of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate in vesicle preparations of the methanogenic bacterium strain Gö1. FEBS Lett 263: 57–60

    Google Scholar 

  • PoirotCM, KengenSWM, ValkE, KeltjensJT, DriftCvan der, VogelsGD (1987) Formation of methylcoenzyme M from formaldehyde by cell-free extracts of Methanobacterium thermoautotrophicum. Evidence for the involvement of a corrinoid-containing methyltransferase. FEMS Microbiol Lett 40: 7–13

    Google Scholar 

  • PolA, DriftCvan der, VogelsGD (1982) Corrinoids from Methanosarcina barkeri: structure of the α-ligand. Biochem Biophys Res Commun 108: 731–737

    Google Scholar 

  • SauerFD (1986) Tetrahydromethanopterin methyltransferase, a component of the methane synthesizing complex of Methanobacterium thermoautotrophicum. Biochem Biophys Res Commun 136: 542–547

    Google Scholar 

  • SauerFD, BlackwellBA, MahadevanS (1986) The role of tetrahydromethanopterin and cytoplasmic cofactor in methane synthesis. Biochem J 235: 453–458

    Google Scholar 

  • SavéantJM, deTacconiN, LexaD, ZicklerJ (1979) Electrochemistry of Vitamin B12. Equilibria, kinetics and mechanisms in the B12a−B12r−B12s oxido-reduction. In: ZagalakB, FriedrichW (eds) Vitamin B12. Walter de Gruyter & Co, Berlin New York, pp 203–212

    Google Scholar 

  • SchönheitP, MollJ, ThauerRK (1980) Growth parameters (Kμmax, Ys) of Methanobacterium thermoautotrophicum. Arch Microbiol 127: 59–65

    Google Scholar 

  • SchulzH, FuchsG (1986) Cobamide-containing membrane protein complex in Methanobacterium. FEBS Lett 198: 279–282

    Google Scholar 

  • SchulzH, AlbrachtSPJ, CoremansJMCC, FuchsG (1988) Purification and some properties of the corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum. Eur J Biochem 171: 589–597

    Google Scholar 

  • SchweigerG, DutschoR, BuckelW (1987) Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron sulfur protein. Eur J Biochem 169: 441–448

    Google Scholar 

  • SmithL (1978) Bacterial cytochromes and their spectral characterization. In: FleischerS, PackerL (eds) Methods in enzymology, vol LIII, cytochromes. Academic Press, New York, pp 202–212

    Google Scholar 

  • StupperichE, JuzaA, EckerskornC, EdelmannL (1990) An immunological study of corrinoid proteins from bacteria revealed homologous antigenic determinants of a soluble corrinoid-dependent methyltransferase and corrinoid-containing membrane proteins from Methanobacterium species. Arch Microbiol 155: 28–34

    Google Scholar 

  • TaylorCD, WolfeRS (1974) A simplified assay for coenzyme M (HSCH2CH2SO3). Resolution of methylcobalamin-coenzyme M methyltransferase and use of sodium borohydride. J Biol Chem 249: 4886–4890

    Google Scholar 

  • ThauerRK (1990) Energy metabolism of methanogenic bacteria. Biochem Biophys Acta 1018: 256–259

    Google Scholar 

  • WijngaardWMHvan de, DriftCvan der, VogelsGD (1988) Involvement of a corrinoid enzyme in methanogenesis from acetate in Methanosarcina barkeri. FEMS Microbiol Lett 52: 165–172

    Google Scholar 

  • WijngaardWMHvan de, LugtigheidRL, DriftCvan der (1991) Reductive activation of the corrinoid-containing enzyme involved in methyl group transfer between methyl-tetrahydromethanopterin and coenzyme M in Methanosarcina barkeri. Antonie van Leeuwenhoek 60: 1–6

    Google Scholar 

  • WoodJM, MouraI, MouraJJG, SantosMH, XavierAV, LeGallJ, ScandellariM (1982) Role of vitamin B12 in methyl transfer for methane biosynthesis by Methanosarcina barkeri. Science 216: 303–305

    Google Scholar 

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Authors and Affiliations

  1. Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität Marburg, Karl-von-Frisch-Strasse, W-3550, Marburg/Lahn, Germany

    R. Fischer, P. Gärtner, A. Yeliseev & R. K. Thauer

  2. Max-Planck-Institut für Terrestrische Mikrobiologie Marburg, Karl-von-Frisch-Strasse, W-3550, Marburg/Lahn, Germany

    R. Fischer, P. Gärtner, A. Yeliseev & R. K. Thauer

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  1. R. Fischer
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  2. P. Gärtner
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  4. R. K. Thauer
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Fischer, R., Gärtner, P., Yeliseev, A. et al. N 5-Methyltetrahydromethanopterin: coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein. Arch. Microbiol. 158, 208–217 (1992). https://doi.org/10.1007/BF00290817

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