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     

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     

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     

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.     

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.     

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     

Kinetic mechanism for the ability of sulfate reducers to out-compete methanogens for acetate

Methanosarcina barkeri and Desulfobacter postgatei are ubiquitous anaerobic bacteria which grow on acetate or acetate plus sulfate, respectively, as sole energy sources. Their apparent K _s values for acetate were determined and found to be approximately 0.2 mM for the sulfate-reducing bacterium and 3 mM for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of acetate consumption by D. postgatei approached 15-fold the rate of M. barkeri at low acetate concentrations. The apparent inhibition of methanogenesis was of the same order as expected from the different K _s value for acetate. Difference in substrate affinities can thus account for the inhibition of methanogenesis from acetate in sulfate-rich environments, where the acetate concentration is well below 1 mM.     

Carbonic anhydrase activity in acetate grown Methanosarcina barkeri

Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K _i=0.5 mM), by azide (apparent K _i=1 mM), and by cyanide (apparent K _i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H₂/CO₂ grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO₂ and CH₄. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO₂ with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).     

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     

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     

Mutagenesis of the C1 oxidation pathway in Methanosarcina barkeri: new insights into the Mtr/Mer bypass pathway

A series of Methanosarcina barkeri mutants lacking the genes encoding the enzymes involved in the C1 oxidation/reduction pathway were constructed. Mutants lacking the methyl-tetrahydromethanopterin (H₄MPT):coenzyme M (CoM) methyltransferase-encoding operon (Δmtr), the methylene-H₄MPT reductase-encoding gene (Δmer), the methylene-H₄MPT dehydrogenase-encoding gene (Δmtd), and the formyl-methanofuran:H₄MPT formyl-transferase-encoding gene (Δftr) all failed to grow using either methanol or H₂/CO₂ as a growth substrate, indicating that there is an absolute requirement for the C1 oxidation/reduction pathway for hydrogenotrophic and methylotrophic methanogenesis. The mutants also failed to grow on acetate, and we suggest that this was due to an inability to generate the reducing equivalents needed for biosynthetic reactions. Despite their lack of growth on methanol, the Δmtr and Δmer mutants were capable of producing methane from this substrate, whereas the Δmtd and Δftr mutants were not. Thus, there is an Mtr/Mer bypass pathway that allows oxidation of methanol to the level of methylene-H₄MPT in M. barkeri. The data further suggested that formaldehyde may be an intermediate in this bypass; however, no methanol dehydrogenase activity was found in Δmtr cell extracts, nor was there an obligate role for the formaldehyde-activating enzyme (Fae), which has been shown to catalyze the condensation of formaldehyde and H₄MPT in vitro. Both the Δmer and Δmtr mutants were able to grow on a combination of methanol plus acetate, but they did so by metabolic pathways that are clearly distinct from each other and from previously characterized methanogenic pathways.     

Isolation and characterization of Glyceraldehyde-3-phosphate dehydrogenase from the common octopus (<Emphasis Type="Italic">Octopus vulgaris </Emphasis>Cuvier, 1797)

The NAD⁺ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately 36 kDa. The Michaelis constants K_m for both NAD⁺ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity V_max of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose.     

Tungstate does not support synthesis of active formylmethanofuran dehydrogenase in Methanosarcina barkeri

Cell extracts of Methanosarcina barkeri grown on methanol in media supplemented with molybdate exhibited a specific activity of formylmethanofuran dehydrogenase of approximately 1 U (1 mol/min)/mg protein. When the growth medium was supplemented with tungstate rather than with molybdate, the specific activity was only 0.04 U/mg. Despite this reduction in specific activity growth on methanol was not inhibited. An inhibition of both growth and synthesis of active formylmethanofuran dehydrogenase was observed, however, when H₂ and CO₂ were the energy substrates. The results indicate that, in contrast to Methanobacterium wolfei and Methanobacterium thermoautotrophicum, M. barkeri possesses only a molybdenum containing formylmethanofuran dehydrogenase and not in addition a tungsten isoenzyme.     

Activities of formylmethanofuran dehydrogenase,methylenetetrahydromethanopterin dehydrogenase,methylenetetrahydromethanopterin reductase,and heterodisulfide reductase in methanogenic bacteria

The activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase were tested in cell extracts of 10 different methanogenic bacteria grown on H₂/CO₂ or on other methanogenic substrates. The four activities were found in all the organisms investigated: Methanobacterium thermoautotrophicum,M. wolfei, Methanobrevibacter arboriphilus, Methanosphaera stadtmanae, Methanosarcina barkeri (strains Fusaro and MS), Methanothrix soehngenii, Methanospirillum hungatei, Methanogenium organophilum, and Methanococcus voltae. Cell extracts of H₂/CO₂ grown M. barkeri and of methanol grown M. barkeri showed the same specific activities suggesting that the four enzymes are of equal importance in CO₂ reduction to methane and in methanol disproportionation to CO₂ and CH₄. In contrast, cell extracts of acetate grown M. barkeri exhibited much lower activities of formylmethanofuran dehydrogenase and methylenetetrahydromethanopterin dehydrogenase suggesting that these two enzymes are not involved in methanogenesis from acetate. In M. stadtmanae, which grows on H₂ and methanol, only heterodisulfide reductase was detected in activities sufficient to account for the in vivo methane formation rate. This finding is consistent with the view that the three other oxidoreductases are not required for methanol reduction to methane with H₂.     

F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification

Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O₂ with H₂ at a maximal specific rate of 0.4 U (mol/min) per mg protein with an apparent K _m for O₂ of 30 M. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a coenzyme F₄₂₀-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F₄₂₀H₂ with 1 O₂ to 2 F₄₂₀ and 2 H₂O. The apparent K _m for F₄₂₀ was 30 M and that for O₂ was 2 M with a V _max of 240 U/mg at 37°C and pH 7.6, the pH optimum of the oxidase. The enzyme did not use NADH or NADPH as electron donor or H₂O₂ as electron acceptor and was not inhibited by cyanide. The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H–X–E–X–D–X₆₂–H–X₁₈–D–X₆₀–H. Sequence comparisons revealed that the F₄₂₀H₂ oxidase from M. arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30–40%), which all have dioxygen reductase activity. With heterologously produced FprA from M. marburgensis it is shown that this protein is also a highly efficient F₄₂₀H₂ oxidase and that it contains 1 FMN and 2 iron atoms. The presence of F₄₂₀H₂ oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions.Dedicated to Hans Schlegel on the occasion of his 80th birthday.     

Tetrahydrofolate-specific enzymes in <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis> and growth dependence of this methanogenic archaeon on folic acid or <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid

Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H₄SPT) rather than tetrahydrofolate (H₄F) as a pterin C₁ carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN, folD, glyA and metF, that are predicted to encode for H₄F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N⁵,N¹⁰-methylene-H₄F dehydrogenase/N⁵,N¹⁰-methenyl-H₄F cyclohydrolase) and GlyA (serine:H₄F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli, purified and found to be specific for methylene-H₄F and H₄F, respectively (apparent K_m below 5 M). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri. The results thus indicate that M. barkeri should contain H₄F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p-aminobenzoic acid, a biosynthetic precursor of H₄F. From the p-aminobenzoic acid requirement, an intracellular H₄F concentration of approximately 5 M was estimated. Evidence is presented that the p-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H₄SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H₄SPT and H₄F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C₁ pools in these methanogens.     

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     

Function of methylcobalamin: coenzyme M methyltransferase isoenzyme II in Methanosarcina barkeri

Methanosarcina barkeri was recently shown to contain two cytoplasmic isoenzymes of methylcobalamin: coenzyme M methyltransferase (methyltransferase 2). Isoenzyme I predominated in methanol-grown cells and isoenzyme II in acetate-grown cells. It was therefore suggested that isoenzyme I functions in methanogenesis from methanol and isoenzyme II in methanogenesis from acetate. We report here that cells of M. barkeri grown on trimethylamine, H₂/CO₂, or acetate contain mainly isoenzyme II. These cells were found to have in common that they can catalyze the formation of methane from trimethylamine and H₂, whereas only acetate-grown cells can mediate the formation of methane from acetate. Methanol-grown cells, which contained only low concentrations of isoenzyme II, were unable to mediate the formation of methane from both trimethylamine and acetate. These and other results suggest that isoenzyme II (i) is employed for methane formation from trimethylamine rather than from acetate, (ii) is constitutively expressed rather than trimethylamine-induced, and (iii) is repressed by methanol. The constitutive expression of isoenzyme II in acetate-grown M. barkeri can explain its presence in these cells. The N-terminal amino acid sequences of isoenzyme I and isoenzyme II were analyzed and found to be only 55% similar.Abbreviations H-S-CoM coenzyme M or 2-mercaptoethane-sulfonate - CH₃-S-CoM methyl-coenzyme M or 2(methylthio)-ethanesulfonate - [Co] cobalamin - CH₃-[Co] methylcobalamin - H₄MPT tetrahydromethanopterin - CH₃-H₄MPT N ⁵-methyltetrahydromethanopterin - MT₁ methyltransferase 1 or methanol: 5-hydroxybenzimidazolyl cobamide methyltransferase - MT₂ methyltransferase 2 or methylcobalamin: coenzyme M methyltransferase - Mops morpholinopropanesulfonate - 1 U = 1 mol/min     

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