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     

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     

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     

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     

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     

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.     

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     

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

From our previous studies on the mechanism of methane formation from acetate it was known that cell extracts of acetate-grown Methanosarcina barkeri (100 000 × g supernatant) catalyze the conversion of acetyl-CoA plus tetrahydromethanopterin (=H₄MPT) to methyl-H₄MPT, CoA, CO₂ and presumably H₂. We report here that these extracts, in the absence of H₄MPT, mediated an isotope exchange between CO₂ ([S]_{0.5 v}=0.2% in the gas phase) and the carbonyl group of acetyl-CoA at almost the same specific rate as the above conversion (10 nmol · min^–1 · mg protein^–1). Both the exchange and the formation of methyl-H₄MPT were inhibited by N₂O, suggesting that a corrinoid could be the primary methyl group acceptor in the acetyl-CoA C-C-cleavage reaction. Both activities were dependent on the presence of H₂ (E⁰=–414 mV). Ti(III)citrate (E⁰=–480 mV) was found to substitute for H₂, indicating a reductive activation of the system. In the presence of Ti(III)citrate it was shown that the formation of CO₂ from the carbonyl group of acetyl-CoA is associated with a 1:1 stoichiometric generation of H₂. Free CO, a possible intermediate in CO₂ and H₂ formation, was not detected.Non-standard abbreviations AcCoA acetyl-CoA - acetyl-P acetyl phosphate - OH-B₁₂ hydroxocobalamin - H-S-CoM coenzyme M = 2-mercaptoethanesulfonate - CH₃-S-CoM methyl-coenzyme M = 2-(methylthio)ethanesulfonate - H-S-HTP N-7-mercaptoheptanoylthreonine phosphate - HTP-S-S-HTP disulfide of H-S-HTP - CoM-S-S-HTP disulfide of H-S-CoM and H-S-HTP - H₄MPT tetrahydromethanopterin - CH₃-H₄MPT N⁵-methyl-H₄MPT - DTT dithiothreitol - MOPS morpholinopropane sulfonic acid     

Formylmethanofuran: tetrahydromethanopterin formyltransferase and N 5,N 10-methylenetetrahydromethanopterin dehydrogenase from the sulfate-reducing Archaeoglobus fulgidus: similarities with the enzymes from methanogenic Archaea

The sulfate-reducing Archaeoglobus fulgidus contains a number of enzymes previously thought to be unique for methanogenic Archaea. The purification and properties of two of these enzymes, of formylmethanofuran: tetrahydromethanopterin formyltransferase and of N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase (coenzyme F₄₂₀ dependent) are described here. A comparison of the N-terminal amino acid sequences and of other molecular properties with those of the respective enzymes from three methanogenic Archaea revealed a high degree of similarity.Abbreviations H₄MPT tetrahydromethanopterin - F₄₂₀ coenzyme - F₄₂₀ formyltransferase, formylmethanofuran: tetrahydromethanopterin formyltransferase - methylene-H₄MPT dehydrogenase N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase - methylene-H₄MPT recductase N ⁵,N ¹⁰-methylenetetrahydromethanopterin reductase - cyclohydrolase N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase - APS adenosine 5-phosphosulfate - MOPS 3-(N-morpholino) propane sulfonic acid - TRICINE N-tris(hydroxymethyl)methylglycine - MES morpholinoethanesulfonic acid - 1 U 1 mol/min     

Carbon monoxide pathway enzyme activities in a thermophilic anaerobic bacterium grown acetogenically and in a syntrophic acetate-oxidizing coculture

We recently isolated an acetate-oxidizing rodshaped eubacterium (AOR) which was capable of oxidizing acetate to CO₂ when grown in coculture with the hydrogenotrophic methanogen Methanobacterium sp. strain THF. The AOR was also capable of growing axenically on H₂CO₂ which it converted to acetate. Previous results for the acetate oxidizing coculture showed isotopic exchange between acetate and CO₂, suggesting that the AOR was using a pathway for acetate oxidation resembling a reveral of the acetogenic (carbon monoxide) pathway. In this study, it was found that production of ¹⁴CO₂ from ¹⁴CH₃COO^- by the coculture was inhibited by 200 M cyanide, while methanogenesis from H₂–CO₂ was unaffected, implying the involvement of carbon monoxide dehydrogenase (CODH) in acetate oxidation. CODH was present at 0.055 mol methyl viologen reduced min^-1 mg^-1 protein in extracts of Methanobacterium sp. strain THF, but was present in higher levels in the acetate oxidizing coculture and in the AOR grown axenically and on H₂–CO₂ (2.0 and 6.4 mol min^-1 mg^-1 protein respectively). Anaerobic activity stains for CODH in native polyacrylamide gels from the AOR coculture showed components co-migrating with bands from both organisms, as well as an additional band in extracts of the coculture. Formate dehydrogenase (FDH) was present in both the AOR coculture and monoculture but not in extracts of H₂–CO₂ grown cells of Methanobacterium sp. strain THF. Formyltetrahydrofolate (FTHF) synthetase was not detectable in extracts of the AOR monoculture or coculture, although it was found in high amounts in extracts of H₂–CO₂ grown cells of the thermophilic acetogen Acetogenium kivui. Extracts of H₂–CO₂ grown cells of the AOR showed a fluorescence spectrum typical of pterin derivatives. Bioassay for folates showed levels to be at anabolic rather than catabolic levels. It is possible that the AOR uses pterins distinct from folate for catabolism. Isocitrate dehydrogenase, a citric acid cycle enzyme, was also present in the AOR, but at anabolic levels and -ketoglutarate dehydrogenase was not detectable.Abbreviations (AOR) acetate-oxidizing rod - (CODH) carbon monoxide dehydrogenase - (FDH) formate dehydrogenase - (FTHF) formyltetrahydrofolate     

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     

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     

One carbon metabolism in methanogenic bacteria

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H₂ oxidation/CO₂ reduction. The optimum temperature for growth and methanogenesis was 37°C. H₂ oxidation proceeded via an F₄₂₀-dependent NADP⁺-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 mol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H₂/CO₂ and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH₄ formed, respectively. During mixotrophic growth on H₂/CO₂ plus methanol, most methane was derived from methanol rather than from CO₂. Similar activities of hydrogenase were observed in cell-free extracts from H₂/CO₂-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO₂, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO₂ and from an organic intermediate more reduced than CO₂. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.Abbreviations CAPS cycloaminopropane sulfonic acid - CH₃-SCoM methyl coenzyme M - DCPIP 2,6-dichlorophenolindophenol - DEAE diethylaminoethyl - dimethyl POPOP 1,4-bis-2-(4-mothyl-5-phenyloxazolyl)-benzene - DNA deoxyribonucleic acid - dpm dismtegrations per min - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - F₄₂₀ factor 420 - G+C guanosine plus cytosine - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - PBBW phosphate buffered basal Weimer - PMS phenazine methosulfate - PPO 2,5-diphenyloxazole - rRNA ribosomal ribonucleic acid - RuBP ribulose-1,5-bisphosphate - Tris tris-hydroxymethyl-aminomethane - max maximum specific growth rate     

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     

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     

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans has been proposed to oxidize acetate to CO₂ via an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway rather than via the citric acid cycle. We report here the presence of the enzyme activities required for the operation of the novel pathway. In cell extracts the following activities were found (values in brackets=specific activities and apparent K _m; 1 U·mg^-1=1 mol·min^-1·mg protein^-1 at 37°C): Acetate kinase (6.3 U·mg^-1; 2 mM acetate; 2.4 mM ATP); phosphate acetyltransferase (60 U·mg^-1, 0.4 mM acetylphosphate; 0.1 mM CoA); carbon monoxide dehydrogenase (29 U·mg^-1; 13% carbon monoxide; 1.3 mM methyl viologen); 5,10-methylenetetrahydrofolate reductase (3 U·mg^-1, 0.06 mM CH₃–FH₄); methylenetetrahydrofolate dehydrogenase (3.6 U·mg^-1, 0.9 mM NAD, 0.1 mM CH₂=FH₄); methenyltetrahydrofolate cyclohydrolase (0.3 U·mg^-1); formyltetrahydrofolate synthetase (3 U·mg^-1, 1.4 mM FH₄, 0.4 mM ATP, 13 mM formate); and formate dehydrogenase (10 U·mg^-1, 0.4 mM formate, 0.5 mM NAD). The specific activities are sufficient to account for the in vivo acetate oxidation rate of 0.26 U·mg^-1.Non-standard abbreviations FH₄ Tetrahydrofolate - CHO-FH₄ N¹⁰-formyltetrahydrofolate - CHFH₄ N⁵,N¹⁰-methenyltetrahydrofolate - CH₂=FH₄ N⁵,N¹⁰-methylenetetrahydrofolate - CH₃–FH₄ N⁵-methyltetrahydrofolate - MOPS morpholinopropane sulfonic acid - DTT d,l-1,4-dithiothreitol - TRIS tris-(hydroxymethyl)-aminomethane - Ap₅A p¹,P⁵-di(adenosine-5)pentaphosphate - MV methyl viologen     

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

The hypothesis of common epitopes in corrinoid-dependent enzymes was tested by a monospecific polyclonal antiserum against the 33 kDa corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum Marburg. Cross-reaction was detected with the 33 kDa and the 31 kDa subunits of the corrinoid-containing enriched 5-methyl-H₄MPT: 5-hydroxybenzimidazolyl cobamide methyltransferase from the cytoplasmic fraction and a 33 kDa protein from the membrane fraction of Methanobacterium thermoauto-trophicum H. This indicates that both proteins have similar antigenic determinants and that they may have similar function as methyltransfer proteins. Also a soluble 20 kDa protein of yet unknown function from Clostridium barkeri cross-reacted with the antiserum. No cross-reactions were observed with the purified corrinoid-containing 2-methyleneglutarate mutase from C. barkeri, the corrinoid/iron-sulfur protein from C. thermoaceticum, the carbon monoxide dehydrogenases from C. thermoaceticum and Methanothrix soehngenii, and the corrinoid-binding protein intrinsic factor from porcine gastric mucosa. Also cell extracts from the corrinoid-rich bacteria Sporomusa ovata, Methanolobus tindarius, Chloroflexus aurantiacus, Propionibacterium shermanii, the membrane fraction and the cytoplasmic fraction of Methanococcus voltae or extracts from human liver, contained no antibody combining sites others than with the preimmunological serum. These findings indicate, that many corrinoid-containing proteins from bacteria have no common antigenic determinants.Abbreviations CH₃-H₄MPT N ⁵-methyl-tetrahydromethanopterin - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - ELISA enzyme linked immunosorbent assay - DSM Deutsche Sammlung von Mikroorganismen     

Reisolation of the carbon monoxide utilizing hydrogen bacterium Pseudomonas carboxydovorans (Kistner) comb. nov.

From enrichment cultures four carbon monoxide utilizing bacteria were isolated; strain OM5 isolated from waste water was studied in detail. The cells are Gram-negative, slightly curved rods, motile by a single subpolarly inserted flagellum. The colonies are smooth, translucent and not slimy.The cells are able to grow autotrophically in mineral medium under an atmosphere of 40% CO, 5% O₂ and 55% N₂ at a doubling time of 20h (30°C) or of 85% H₂, 5% O₂ and 10% CO₂ at a doubling time of 7h. Heterotrophic growth occurrd on organic acids such as acetate (t _d =8h), pyruvate (t _d =8h), lactate, crotonate, malate, succinate (t _d =8h), formate (t _d =35h) and glyoxylate as substrates.The enzyme system for carbon monoxide utilization is formed only during growth on CO; hydrogenase is present in cells grown on CO or on H₂+CO₂ as well as grown on pyruvate. The rate of oxygen reduction by intact CO-grown cells is 3.7-fold higher in the presence of hydrogen than in the presence of carbon monoxide. During growth the stoichiometry of gas uptake was 6.1 CO+2.8 O₂+H₂O CH₂O+5.1 CO₂. For the new isolate the name Pseudomonas carboxydovorans (Kistner) comb. nov. has been proposed.Part of this work was presented at the Second International Symposium on Microbial Growth on C₁-Compounds in Puschino, USSR, September 12th–16th, 1977.     

Energetics of the growth of Methanobacterium thermoautotrophicum and Methanococcus thermolithotrophicus on ammonium chloride and dinitrogen

Methanobacterium thermoautotrophicum was grown in continuous culture in a fermenter gassed with H₂ and CO₂ as sole carbon and energy sources, and in a medium which contained either NH₄Cl or gaseous N₂ as nitrogen source. Growth was possible with N₂. Steady states were obtained at various gas flow rates with NH₄Cl and with and the maintenance coefficient varied with the gas input and with the nitrogen source. Growth of Methanococcus thermolithotrophicus in continuous culture in a fermenter gassed with H₂, CO₂ as nitrogen, carbon and energy sources was also examined.Abbreviations molecular growth yield (g dry weight of cells per mol of CH₄ evolved) - growth rate (h^-1) - D dilution rate (h^-1) - rate (h_-1); relation of Neijssel and Tempest and of Stouthamer and Bettenhaussen - energy     

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.