Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two -, - and -subunits and that it contained the nickel porphinoid coenzyme F₄₃₀ as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the -subunit was determined. A comparison with the N-terminal sequences of the -subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity.Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO₂ (apparent V_max at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N ⁵,N¹⁰-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (H₂-forming), 33 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin reductase (coenzyme F₄₂₀ dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F₄₂₀-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent K_m values for these enzymes and the effect of salts on their activities were determined.The coenzyme F₄₂₀ present in M. kandleri was identified as coenzyme F₄₂₀-2 with 2 -glutamyl residues.Abbreviations H–S-CoM coenzyme M - CH₃–S-CoM methylcoenzyme M - H–S-HTP 7-mercaptoheptanoylthreonine phosphate - MFR methanofuran - CHO-MFR formyl-MFR - H₄MPT tetrahydromethanopterin - CHO–H₄MPT N ⁵-formyl-H₄MPT - CH=H₄MPT⁺ N ⁵,N¹⁰-methenyl-H₄MPT - CH₂=H₄MPT N ⁵,N¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - 1 U= 1 mol/min     

Function of methanofuran,tetrahydromethanopterin, and coenzyme F420 in Archaeoglobus fulgidus

Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO₂ and H₂S. The organism contains coenzyme F₄₂₀, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F₄₂₀ oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO₂ via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C₁-intermediates otherwise only used by methanogenic bacteria.Non-standard abbreviations APS adenosine 5-phosphosulfate - BV benzyl viologen - DCPIP 2,6-dichlorophenolindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - H₄F tetrahydrofolate - H₄MPT tetrahydromethanopterin - CH₂ H₄MPT, methylene-H₄MPT - CH H₄MPT, methenyl-H₄MPT - Mes morpholinoethane sulfonic acid - MFR methanofuran - Mops morpholinopropane sulfonic acid - MV methyl viologen - Tricine N-tris(hydroxymethyl)-methylglycine - U mol product formed per min     

N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus

Methylene-H₄MPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F₄₂₀ as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a K _m for methylene-H₄MPT of 16 M, a K _m for F₄₂₀H₂ of 4 M, and a V _max of 450 U/mg (K_cat=265 s^-1) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F₄₂₀ (0.2 mM), methylene-H₄MPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-H₄MPT reductase (F₄₂₀-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described.Abbreviations H₄MPT tetrahydromethanopterin - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - CHH₄MPT methenyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formyl-MFR - 1 U 1 mol/min     

A F420-dependent NADP reductase in the extremely thermophilic sulfate-reducing Archaeoglobus fulgidus

Archaeoglobus fulgidus, a sulfate-reducing Archaeon with a growth temperature optimum of 83°C, uses the 5-deazaflavin coenzyme F₄₂₀ rather than pyridine nucleotides in catabolic redox processes. The organism does, however, require reduced pyridine nuclcotides for biosynthetic purposes. We describe here that the Archaeon contains a coenzyme F₄₂₀-dependent NADP reductase which links anabolism to catabolism. The highly thermostable enzyme was purfied 3600-fold by affinity chromatography to apparent homogeneity in a 60% yield. The native enzyme with an apparent molecular mass of 55 kDa was composed of only one type of subunit of apparent molecular mass of 28 kDa. Spectroscopic analysis of the enzyme did not reveal the presence of any chromophoric prosthetic group. The purified enzyme catalyzed the reversible reduction of NADP (apparent K _M 40 M) with reduced F₄₂₀ (apparent K _M 20M) with a specific activity of 660 U/mg (apparent V _max) at pH 8.0 (pH optimum) and 80°C (temperature optimum). It was specific for both coenzyme F₄₂₀ and NADP. Sterochemical investigations showed that the F₄₂₀-dependent NADP reductase was Si face specific with respect to C5 of F₄₂₀ and Si face specific with respect to C4 of NADP.Abbreviations F₄₂₀ coenzyme F₄₂₀ - F₄₂₀H₂ 1,5-dihydrocoenzyme F₄₂₀ - H₄MPT tetrahydromethanopterin - CH=H₄MPT N⁵, N¹⁰-methylenetetrahydromethanopterin - MFR methanofuran - HPLC high performance liquid chromatography - methylene-H₄MPT dehydrogenase N⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase - 1 U = 1 mol/min     

Two N 5, N 10-methylenetetrahydromethanopterin dehydrogenases in the extreme thermophile Methanopyrus kandleri: characterization of the coenzyme F420-dependent enzyme

It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H₂-forming N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor. We describe here the presence in this Archaeon of a second N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F₄₂₀-dependent. This enzyme was purified and characterized. The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7±0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa. The enzyme activity increased to an optimum with increasing salt concentrations. Optimal salt concentrations were e.g. 2 M (NH₄)₂SO₄, 2 M Na₂HPO₄, 1.5 M K₂HPO₄, and 2 M NaCl. In the absence of salts the enzyme exhibited almost no activity. The salts affected mainly the V _max rather than the K _m of the enzyme. The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group. In the presence of M (NH₄)₂SO₄ the V _max was 4000 U/mg (k _cat=2400 s^-1) and the K _m for N ⁵,N ¹⁰-methylenetetrahydromethanopterin and for coenzyme F₄₂₀ were 80 M and 20 M, respectively. The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM K₂HPO₄ at 90°C for one hour. The N-terminal amino acid sequence was found to be similar to that of the F₄₂₀-dependent N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus.Abbreviations H₄MPT tetrahydromethanopterin - F₄₂₀ coenzyme F₄₂₀ - CH₂=H₄MPT N ⁵,N ¹⁰-methylenetrahydromethanopterin - CHH₄MPT⁺ N ⁵,N ¹⁰-methenyltetrahydromethanopterin - methylene-H₄MPT dehydrogenase N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase - Mops N-morpholinopropane sulfonic acid - Tricine N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min     

Enzymes and coenzymes of the carbon monoxide dehydrogenase pathway for autotrophic CO2 fixation in Archaeoglobus lithotrophicus and the lack of carbon monoxide dehydrogenase in the heterotrophic A. profundus

Archaeoglobus lithotrophicus is a hyperthermophilic Archaeon that grows on H₂ and sulfate as energy sources and CO₂ as sole carbon source. The autotrophic sulfate reducer was shown to contain all the enzyme activities and coenzymes of the reductive carbon monoxide dehydrogenase pathway for autotrophic CO₂ fixation as operative in methanogenic Archaea. With the exception of carbon monoxide dehydrogenase these enzymes and coenzymes were also found in A. profundus. This organism grows lithotrophically on H₂ and sulfate, but differs from A. lithotrophicus in that it cannot grow autotrophically: A. profundus requires acetate and CO₂ for biosynthesis. The absence of carbon monoxide dehydrogenase in A. profundus is substantiated by the observation that this organism, in contrast to A. lithotrophicus, is not mini-methanogenic and contains only relatively low concentrations of corrinoids.Abbreviations F ₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formylmethanofuran - H ₄MPT 5,6,7,8-tetrahydromethanopterin - CHO–H ₄MPT N⁵ formyl-H₄MPT - CHH₄MPT⁺N⁵ methenyl-H₄MPT - CH ₂=H₄MPT N⁵, N¹⁰ methylene-H₄MPT - CH ₃–H₄MPT N⁵ methyl-H₄MPT - H ₄F tetrahydrofolate - I U 1 mol/min - t _d doubling time     

Purification,properties and primary structure of H2-formingN 5,N10-methylenetetrahydromethanopterin dehydrogenase fromMethanococcus thermolithotrophicus

H₂-FormingN ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (Hmd) is a novel type of hydrogenase found in methanogenic Achaea that contains neither nickel nor iron-sulfur clusters. The enzyme has previously been characterized fromMethanobacterium thermoautotrophicum and fromMethanopyrus kandleri. We report here on the purification and properties of the enzyme fromMethanococcus thermolithotrophicus. Thehmd gene was cloned and sequenced. The results indicate that the enzyme fromMc. thermolithotrophicus is functionally and structurally closely related to the H₂-forming methylene tetrahydromethanopterin dehydrogenase fromMb. thermoautotrophicum andMp. kandleri. From amino acid sequence comparisons of the three enzymes, a phylogenetic tree was deduced that shows branching orders similar to those derived from sequence comparisons of the 16S rRNA of the orders Methanococcales, Methanobacteriales, and Methanopyrales.Abbreviations H ₂ Forming dehydrogenase orHmd - H₂-FormingN ⁵,N¹⁰ methylene tetrahydromethanopterin dehydrogenase - H ₄MPT Tetrahydromethanopterin - CH ₂=H₄MPT N⁵,N¹⁰ Methylene tetrahydromethanopterin - CHH ₄MPT⁺ N⁵,N¹⁰ Methenyltetrahydromethanopterin - MALDI-TOF-MS Matrix-assisted laser desorption     

Effect of methanogenic substrates on coenzyme F420-dependent N5,N10-methylene-H4MPT dehydrogenase,N5,N10-methenyl-H4MPT cyclohydrolase and F420-reducing hydrogenase activities in Methanosarcina barkeri

We measured F₄₂₀-dependent N⁵,N¹⁰-methylenetetrahydro-methanopterin dehydrogenase, N⁵, N¹⁰-methenyltetrahydro-methanopterin cyclohydrolase, and F₄₂₀-reducing hydrogenase levels in Methanosarcina barkeri grown on various substrates. Variation in dehydrogenase levels during growth on a specific substrate was usually <3-fold, and much less for cyclohydrolase. H₂–CO₂-, methanol-, and H₂–CO₂+ methanol-grown cells had roughly equivalent levels of dehydrogenase and cyclohydrolase. In acetate-grown cells cyclohydrolase level was lowered 2 to 3-fold and dehydrogenase 10 to 80-fold; this was not due to repression by acetate, since, if cultures growing on acetate were supplemented with methanol or H₂–CO₂, dehydrogenase levels increased 14 to 19-fold, and cyclohydrolase levels by 3 to 4-fold. Compared to H₂–CO₂- or methanol-grown cells, acetate-or H₂–CO₂ + methanol-grown cells had lower levels of and less growth phase-dependent variation in hydrogenase activity. Our data are consistent with the following hypotheses: 1. M. barkeri oxidizes methanol via a portion of the CO₂-reduction pathway operated in the reverse direction. 2. When steps from CO₂ to CH₃-S-CoM in the CO₂-reduction pathway (in either direction) are not used for methanogenesis, hydrogenase activity is lowered.Abbreviations MF methanofuran - H₄MPT 5,6,7,8-tetrahydromethanopterin - HS-HTP 7-mercaptoheptanoylthreonine phosphate - CoM-S-S-HTP heterodisulfide of HS-CoM and HS-HTP - F₄₂₀ coenzyme F₄₂₀ (a 7,8-didemethyl-8-hydroxy-5-deaza-riboflavin derivative) - H₂F₄₂₀ reduced coenzyme F₄₂₀ - HC⁺=H₄MPT N⁵,N¹⁰-methenyl-H₄MPT - H₂C=H₄MPT N⁵,N¹⁰-methylene-H₄MPT - H₃C=H₄MPT N⁵-methyl-H₄MPT - BES 2-bromoethanesulfonic acid     

Pathways of autotrophic CO2 fixation and of dissimilatory nitrate reduction to N2O in Ferroglobus placidus

The strictly anaerobic Archaeon Ferroglobus placidus was grown chemolithoautotrophically on H₂ and nitrate and analyzed for enzymes and coenzymes possibly involved in autotrophic CO₂ fixation. The following enzymes were found [values in parentheses = μmol min^–1 (mg protein)^–1]: formylmethanofuran dehydrogenase (0.2), formylmethanofuran:tetrahydromethanopterin formyltransferase (0.6), methenyltetrahydromethanopterin cyclohydrolase (10), F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase (1.5), F₄₂₀-dependent methylenetetrahydromethanopterin reductase (0.4), and carbon monoxide dehydrogenase (0.1). The cells contained coenzyme F₄₂₀ (0.4 nmol/mg protein), tetrahydromethanopterin (0.9 nmol/ mg protein), and cytochrome b (4 nmol/mg membrane protein). From the enzyme and coenzyme composition of the cells, we deduced that autotrophic CO₂ fixation in F. placidus proceeds via the carbon monoxide dehydrogenase pathway as in autotrophically growing Archaeoglobus and Methanoarchaea species. Evidence is also presented that cell extracts of F. placidus catalyze the reduction of two molecules of nitrite to 1 N₂O with NO as intermediate (0.1 μmol N₂O formed per min and mg protein), showing that – at least in principle –F. placidus has a denitrifying capacity. Received: 23 August 1996 / Accepted: 6 November 1996     

N 5,N 10-Methenyltetrahydromethanopterin cyclohydrolase from the extremely thermophilic sulfate reducing Archaeoglobus fulgidus: comparison of its properties with those of the cyclohydrolase from the extremely thermophilic Methanopyrus kandleri

Archaeoglobus fulgidus and Methanopyrus kandleri are both extremely thermophilic Archaea with a growth temperature optimum at 83°C and 98°C, respectively. Both Archaea contain an active N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase. The enzyme from M. kandleri has recently been characterized. We describe here the purification and properties of the enzyme from A. fulgidus.The cyclohydrolase from A. fulgidus was purified 180-fold to apparent homogeneity and its properties were compared with those recently published for the cyclohydrolase from M. kandleri. The two cytoplasmic enzymes were found to have very similar molecular and catalytic properties. They differed, however, significantly with respect of the effect of K₂HPO₄ and of other salts on the activity and the stability. The cyclohydrolase from A. fulgidus required relatively high concentrations of K₂HPO₄ (1 M) for optimal thermostability at 90°C but did not require salts for activity. Vice versa, the enzyme from M. kandleri was dependent on high K₂HPO₄ concentrations (1.5 M) for optimal activity but not for thermostability. Thus the activity and structural stability of the two thermophilic enzymes depend in a completely different way on the concentration of inorganic salts. The molecular basis for these differences are discussed.Abbreviations H₄MPT tetrahydromethanopterin - MFR methanofuran - CH₃–H₄MPT N ⁵-methyl-H₄MPT - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₂H₄MPT N ⁵,N ¹⁰-methenyl-H₄MPT - CHO–H₄MPT N ⁵ formyl-H₄MPT - CHO-MFR formyl-MFR - cyclohydrolase N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase - MOPS 3-(N-morpholino) propane sulfonic acid - TRICINE N-tris (hydroxymethyl) methyl glycine - 1 U=1 mol/min     

Purification and properties of N 5 ,N 10 -methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) from the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogens known so far. N ⁵ N ¹⁰ -Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogenesis from CO₂, was purified from this hyperthermophile. The apparent molecular mass of the native enzyme was found to be 300 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only one polypeptide of apparent molecular mass 38 kDa. The ultraviolet/visible spectrum of the enzyme was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was specific for reduced coenzyme F₄₂₀ as electron donor; NADH, NADPH or reduced dyes could not substitute for the 5-deazaflavin. The catalytic mechanism was found to be of the ternary complex type as deduced from initial velocity plots. V _max at 65°C and pH 6.8 was 435 U/mg (k_cat=275 s^-1) and the K _m for methylenetetrahydro-methanopterin and for reduced F₄₂₀ were 6 M and 4 M, respectively. From Arrhenius plots an activation energy of 34 kJ/mol was determined. The Q ₁₀ between 40°C and 90°C was 1.5.The reductase activity was found to be stimulated over 100-fold by sulfate and by phosphate. Maximal stimulation (100-fold) was observed at a sulfate concentration of 2.2 M and at a phosphate concentration of 2.5 M. Sodium-, potassium-, and ammonium salts of these anions were equally effective. Chloride, however, could not substitute for sulfate or phosphate in stimulating the enzyme activity.The thermostability of the reductase was found to be very low in the absence of salts. In their presence, however, the reductase was highly thermostable. Salt concentrations between 0.1 M and 1.5 M were required for maximal stability. Potassium salts proved more effective than ammonium salts, and the latter more effective than sodium salts in stabilizing the enzyme activity. The anion was of less importance.The N-terminal amino acid sequence of the reductase from M. kandleri was determined and compared with that of the enzyme from Methanobacterium thermoautotrophicum and Methanosarcina barkeri. Significant similarity was found.Abbreviations H₄MPT tetrahydromethanopterin - CH₂=H₄MPT N ⁵ ,N ¹⁰ -methylene-H₄MPT - CH₃-H₄MPT N ⁵-methyl-H₄MPT - CHH₄MPT⁺ N ⁵ ,N ¹⁰ -methenyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀; 1 U=1 mol/min     

Coenzyme F420 dependent N5, N10-methylenetetrahydromethanopterin dehydrogenase in methanol grown Methanosarcina barkeri

The dehydrogenation of N ⁵,N ¹⁰-methylenetetrahydromethanopterin (CH₂=H₄MPT) to N ⁵,N ¹⁰-methenyltetrahydromethanopterin (CH≡H₄MPT⁺) is an intermediate step in the oxidation of methanol to CO₂ in Methanosarcina barkeri. The reaction is catalyzed by CH₂=H₄MPT dehydrogenase, which was found to be specific for coenzyme F₄₂₀ as electron acceptor; neither NAD, NADP nor viologen dyes could substitute for the 5-deazaflavin. The dehydrogenase was anaerobically purified almost 90-fold to apparent homogeneity in a 32% yield by anion exchange chromatography on DEAE Sepharose and Mono Q HR, and by affinity chromatography on Blue Sepharose. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band with an apparent mass of 31 kDa. The apparent molecular mass of the native enzyme determined by polyacrylamide gradient gel electrophoresis was 240 kDa. The ultraviolet/visible spectrum of the purified enzyme was almost identical to that of albumin suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentrations were linear: the apparent K _m for CH₂=H₄MPT and for coenzyme F₄₂₀ were found to be 6 μM and 25 μM, respectively. V_max was 4,000 μmol min^-1·mg^-1 protein (k_cat=2,066 s^-1) at pH 6 (the pH optimum) and 37°C. The Arrhenius activation energy was 40 kJ/mol. The N-terminal amino acid sequence was found to be 50% identical with that of the F₄₂₀-dependent CH₂=H₄MPT dehydrogenase isolated from H₂/CO₂ grown Methanobacterium thermoautotrophicum.     

The role of formylmethanofuran: tetrahydromethanopterin formyltransferase in methanogenesis from carbon dioxide

Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified to electrophoretic homogeneity from cells of Methanobacterium thermoautotrophicum. The enzyme is a tetramer of similar or identical subunits (Mr = 41,000). The equilibrium favors transfer of the formyl group to tetrahydromethanopterin (H4MPT) at physiological pH. The product of formyl transfer by the purified enzyme was shown by a number of criteria to be 5-formyl-H4MPT, as opposed to 10-formyl-H4MPT or 5,10-methenyl-H4MPT. Reconstitution of a portion of the methanogenic C1 cycle was effected by combining purified formyltransferase, methenyl-H4MPT cyclohydrolase, formylmethanofuran, and H4MPT to give methenyl-H4MPT. Additional reconstitution experiments established that the formyltransferase is an essential enzyme for the conversion of carbon dioxide to methane. In conjunction with previously published data (Donnelly, M.I., Escalante-Semerena, J.C., Rinehart, K. L., Jr., and Wolfe, R.S. (1985) Arch. Biochem. Biophys. 242, 430-439), these data substantiate the role of 5-formyl-H4MPT as an intermediate of methanogenesis.     

N 5,N 10-Methenyltetrahydromethanopterin cyclohydrolase from the extreme thermophile Methanopyrus kandleri: increase of catalytic efficiency (kcat/K M) and thermostability in the presence of salts

The activity of purified N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase from Methanopyrus kandleri was found to increase up to 200-fold when potassium phosphate was added in high concentrations (1.5 M) to the assay. A 200-fold stimulation was also observed with sodium phosphate (1 M) and sodium sulfate (1 M) whereas stimulation by potassium sulfate (0.8 M), ammonium sulfate (1.5 M), potassium chloride (2.5 M), and sodium chloride (2 M) was maximal 100-fold. A detailed kinetic analysis of the effect of potassium phosphate revealed that this salt exerted its stimulatory effect by decreasing the K _m for N ⁵,N ¹⁰-methenyltetrahydromethanopterin from 2 mM to 40 M and by increasing the V _max from 2000 U/mg (k_cat=1385 s^-1) to 13300 U/mg (k_cat=9200 s^-1). Besides increasing the catalytic efficiency (k_cat/K _m) salts were found to protect the cyclohydrolase from heat inactivation. For maximal thermostability much lower concentrations (0.1 M) of salts were required than for maximal activity.Abbreviations H₄MPT tetrahydromethanopterin - N ⁵,N ¹⁰-methenyl-H₄MPT - CHO-H₄MPT N ⁵-formyl-H₄-MPT - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄-MPT N ⁵-methyl-H₄MPT - MOPS -N-morpholinopropane sulfonic acid - TRICINE N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO₂ reduction to CH₄ contain either an NADP-dependent or a coenzyme F₄₂₀-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase and the N ⁵, N ¹⁰-methylenetetrahydromethanopterin reductase, two enzymes involved in CO₂ reduction to CH₄, are specific for F₄₂₀. This raised the question how F₄₂₀H₂ is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F₄₂₀ with NADPH. The F₄₂₀-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced. Received: 12 May 1997 / Accepted: 1 July 1997     

Function of H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum in coenzyme F420 reduction with H2

Summary In most methanogenic archaea, two hydrogenase systems that can catalyze the reduction of coenzyme F₄₂₀ (F₄₂₀) with H₂ are present: (1) the F₄₂₀-reducing hydrogenase, which is a nickel iron-sulfur flavoprotein composed of three different subunits, and (2) the N ⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase system, which is composed of H₂-forming methylenetetrahydromethanopterin dehydrogenase and F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase, both metal-free proteins without an apparent prosthetic group. We report here that in nickel-limited chemostat cultures of Methanobacterium thermoautotrophicum, the specific activity of the F₄₂₀-reducing Ni/Fe-hydrogenase was essentially zero, whereas that of the H₂-forming methylenetetrahydromethanopterin dehydrogenase was six times higher, and that of the F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase was four times higher than in cells grown under non-nickel-limited conditions. This evidence supports the hypothesis that when M. thermoautotrophicum grows under conditions of nickel limitation, the reduction of F₄₂₀ with H₂ is catalyzed by the metal-free methylenetetrahydromethanopterin dehydrogenase system. Received: 9 September 1997 / Accepted: 30 October 1997     

The structure of formylmethanofuran: tetrahydromethanopterin formyltransferase in complex with its coenzymes

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 (H4MPT) 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 3A that close the H4MPT 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 N5 atom of tetrahydromethanopterin onto the formyl carbon atom of formylmethanofuran.     

Methyltetrahydromethanopterin as an intermediate in methanogenesis from acetate in Methanosarcina barkeri

Cell extracts (100,000×g) of acetate grown Methanosarcina barkeri (strain MS) catalyzed CH₄ and CO₂ formation from acetyl-CoA with specific activities of 50 nmol·min^-1·mg protein^-1. CH₄ formation was found to be dependent on tetrahydromethanopterin (H₄MPT) (apparent K _M=4 μM), coenzyme M (H-S-CoM), and 7-mercaptoheptanoylthreonine phosphate (H-S-HTP=component B) rather than on methanofuran (MFR) and coenzyme F₄₂₀ (F₄₂₀). Methyl-H₄MPT was identified as an intermediate. This compound accumulated when H-S-CoM and H-S-HTP were omitted from the assays. These and previous results indicate that methanogenesis from acetate proceeds via acetyl phosphate, acetyl-CoA, methyl-H₄MPT, and CH₃-S-CoM as intermediates. The disproportionation of formaldehyde to CO₂ and CH₄ was also studied. This reaction was shown to be dependent on H₄MPT, MFR, F₄₂₀, H-S-CoM, and H-S-HTP.     

Interconversion of F430 derivatives of methanogenic bacteria

F₄₃₀ is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F₄₃₀ is thermolabile and in the presence of acetonitrile or C10 _in4 ^sup- two epimerization products are obtained upon heating; in the absence of these compounds F₄₃₀ is oxidized to 12, 13-didehydro-F₄₃₀. The latter is stereoselectively reduced under H₂ atmosphere to F₄₃₀ by cell-free extracts of M. barkeri or M. thermoautotrophicum. H₂ may be replaced by the reduced methanogenic electron carrier coenzyme F₄₂₀.Abbreviations CH₃S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F₄₃₀ Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F₄₃₀ and 12,13-di-epi-F₄₃₀ the 12, 13- and 12, 13-derivatives of F₄₃₀ - 12, 13-didehydro-F₄₃₀ F₄₃₀ oxidized at C-12 and C-13 - coenzyme F₄₂₀ 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F₄₂₀H₂ reduced coenzyme F₄₂₀ - MV⁺ methylviologen semiquinone - HPLC high-performance liquid chromatography