H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum. Studies of the catalytic mechanism of H2 formation using hydrogen isotopes.

H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase is a novel hydrogenase found in most methanogenic archaea. It catalyzes the reversible conversion of N5,N10-methylenetetrahydromethanopterin (CH2 = H4MPT) to N5,N10-methenyltetrahydromethanopterin (CH identical to H4MPT+) and dihydrogen; CH2 = H4MPT + H+<-->CH identical to H4MPT(+) + H2; delta G degrees ' = + 5 kJ/mol. In the following investigation, the formation of H2, HD and D2 was studied in experiments in which either the methylene group of CH2 = H4MPT or water were deuterium labelled. In the case of CD2 = H4MPT and H2O, the dihydrogen formed immediately after the start of the reaction was composed of approximately 50% HD and 50% of H2 at all pH tested. In the case of CH2 = H4MPT and D2O, the dihydrogen generated was composed of approximately 50% HD and 50% D2 at pD 5.7 and of approximately 85% HD and 15% D2 at pD 7.0. Evidence is presented that the enzyme catalyzes a CH identical to H4MPT(+)-dependent isotopic exchange between HD and H2O and between HD and D2O, yielding H2 and D2, respectively. A catalytic mechanism aimed to explain these findings is discussed.

Determination of the relative configuration of 5,6,7,8-tetrahydromethanopterin by two-dimensional NMR spectroscopy.

The relative configuration of the pterin moiety of 5,6,7,8-tetrahydromethanopterin 1, a coenzyme isolated from methanogenic archaea, has been determined by two-dimensional NMR spectroscopy of N5,N10-methenyl-5,6,7,8-tetrahydromethanopterin 2 to be rel-(6R; 7S; 11R). The complete proton resonance assignment of the pterin moiety of N5,N10-methylene-5,6,7,8-tetrahydromethanopterin 3 is described including the relative stereospecific assignment of the C(14a) methylene protons.

H2-forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron-sulfur clusters in methanogenic archaea.

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2 = H4MPT) to methenyltetrahydromethanopterin (CH identical to H4MPT+) and H2 and was therefore named H2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H2 or CH2 = H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (Vmax) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between 3H2 and 1H+; (d) the enzyme catalyzed the reduction of CH identical to H4MPT+ with 3H2 generating [methylene-3H]CH2 = H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxin-type iron-sulfur protein. Properties of the H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.

Hydrogen-forming and coenzyme-F420-reducing methylene tetrahydromethanopterin dehydrogenase are genetically distinct enzymes in Methanobacterium thermoautotrophicum (Marburg).

A coenzyme-F420-reducing and an H2-forming methylenetetrahydromethanopterin dehydrogenase have been isolated from Methanobacterium thermoautotrophicum (Marburg). Indirect evidence suggested that the former enzyme (32 kDa) might be derived from the latter enzyme (42 kDa) by proteolysis. To test this hypothesis the gene sequence of the H2-forming dehydrogenase was determined and compared with the N-terminal amino acid sequence of the F420-reducing dehydrogenase. No corresponding sequences were found indicating that the two dehydrogenases are genetically distinct enzymes. With purified enzyme preparations it is shown that the activity of the F420-reducing dehydrogenase is inhibited in the presence of the H2-forming enzyme. This finding is discussed in terms of substrate competition.

N5, N10-methylenetetrahydromethanopterin dehydrogenase (H2-forming) from the extreme thermophile Methanopyrus kandleri.

Methanopyrus kandleri is a novel abyssal methanogenic archaebacterium growing at 110 degrees C on H2 and CO2. The N5, N10-methylenetetrahydromethanopterin dehydrogenase, an enzyme involved in methanogenesis from CO2 and H2, was purified from this hyperthermophile and characterized. The dehydrogenase was found to be composed of only one polypeptide of apparent molecular mass 44 kDa. The UV/Vis spectrum was similar to that of albumin. The protein catalyzed the reversible dehydrogenation of N5, N10-methylenetetrahydromethanopterin (CH2 = H4MPT) to N5, N10-methenyltetrahydromethanopterin (CH identical to H4MPT+) and molecular hydrogen: CH2 = H4MPT H+ in equilibrium CH identical to H4MPT+ +H2. The rate of CH2 = H4MPT dehydrogenation (apparent Vmax) at 65 degrees C and pH 5.8 was 1500 U/mg, the apparent Km for CH2 = H4MPT was 50 microM, the Arrhenius activation energy was 52 kJ/mol, and the Q10 between 30 degrees C and 70 degrees C was 2.0. The specific activity increased hyperbolically with the proton concentration between pH 7 and pH 4.5. The purified dehydrogenase did not catalyze the reduction of viologen dyes, of coenzyme F420, and of pyridine nucleotides with either CH2 = H4MPT or H2. For activity the CH2 = H4MPT dehydrogenase required the presence of salts. Fifty percent of maximal activity was reached at salt concentrations of 100 mM, potassium phosphate, potassium chloride, and sodium chloride being almost equally effective in stimulating the enzyme activity. Cell extracts of M. kandleri did not loose CH2 = H4MPT dehydrogenase activity when incubated at 90 degrees C for 60 min. The purified enzyme, however, proved very thermolabile. The purified enzyme, however, proved very thermolabile.(ABSTRACT TRUNCATED AT 250 WORDS)

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 degrees 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 alpha-, beta- and gamma-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 gamma-subunit was determined. A comparison with the N-terminal sequences of the gamma-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 Vmax at 65 degrees C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran:tetrahydro-methanopterin formyltransferase, 13 U/mg; N5,N10-methylenetetrahydromethanopterin cyclohydrolase, 14U/mg; N5,N10-methenyltetrahydromethanopterin dehydrogenase (H2-forming), 33 U/mg; N5,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 Km 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-gamma-glutamyl residues.