Publications

• Purification and properties of 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri.

  te Brommelstroet B.W., Hensgens C.M., Geerts W.J., Keltjens J.T., van der Drift C., Vogels G.D.

  The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri was purified 313-fold to a specific activity of 470 mumol min-1 mg-1 at 37 degrees C and pH 7.8. At this stage, the enzyme was pure as judged from polyacrylamide gel electrophoresis. The monofunctional enzyme was o ... >> More

  The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri was purified 313-fold to a specific activity of 470 mumol min-1 mg-1 at 37 degrees C and pH 7.8. At this stage, the enzyme was pure as judged from polyacrylamide gel electrophoresis. The monofunctional enzyme was oxygen stable, but the presence of a detergent proved to be essential for its stability. Like the cyclohydrolase purified from Methanobacterium thermoautotrophicum (A. A. Dimarco, M. I. Donnelly, and R. S. Wolfe, J. Bacteriol. 168:1372-1377, 1986), the protein showed an apparent Mr of 82,000, and it is composed of two identical subunits as was concluded from nondenaturating and denaturating polyacrylamide gel electrophoresis. The enzymes from M. thermoautotrophicum and M. barkeri markedly differ with respect to the hydrolysis product of 5,10-methenyltetrahydromethanopterin: 5-formyl- and 10-formyltetrahydromethanopterin, respectively. The apparent Km for 5,10-methenyltetrahydromethanopterin was 0.57 mM at 37 degrees C and pH 7.8. << Less

  J Bacteriol 172:564-571(1990) [PubMed] [EuropePMC]

• Si-face stereospecificity at C5 of coenzyme F420 for F420-dependent N5,N10-methylenetetrahydromethanopterin dehydrogenase, F420-dependent N5,N10-methylenetetrahydromethanopterin reductase and F420H2:dimethylnaphthoquinone oxidoreductase.

  Kunow J., Schworer B., Setzke E., Thauer R.K.

  Coenzyme F420-dependent enzymes catalyze the reversible reduction of F420 by stereospecific hydride transfer to C5 of 5-deazaflavin. Two F420-dependent enzymes have been investigated with respect to the stereochemistry of hydride transfer, the F420-dependent NADP reductase and the F420-reducing hy ... >> More

  Coenzyme F420-dependent enzymes catalyze the reversible reduction of F420 by stereospecific hydride transfer to C5 of 5-deazaflavin. Two F420-dependent enzymes have been investigated with respect to the stereochemistry of hydride transfer, the F420-dependent NADP reductase and the F420-reducing hydrogenase. Both enzymes were found to be Si-face specific. In this study we report that three additional F420-dependent enzymes are also Si-face specific: N5,N10-methylenetetrahydromethanopterin dehydrogenase, N5,N10-methylenetetrahydromethanopterin reductase and coenzyme F420H2: dimethylnaphthoquinone oxidoreductase (F420H2 dehydrogenase). Thus, all five characterized F420-dependent enzymes are Si-face specific, which is noteworthy since coenzyme F420 is functionally similar to pyridine nucleotides and both Si-face specific and Re-face specific pyridine-nucleotide-dependent enzymes exist. << Less

  Eur J Biochem 214:641-646(1993) [PubMed] [EuropePMC]

• Purification and properties of 5,10-methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase, two coenzyme F420-dependent enzymes, from Methanosarcina barkeri.

  te Brommelstroet B.W., Geerts W.J., Keltjens J.T., van der Drift C., Vogels G.D.

  5,10-Methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase have been purified to homogeneity by a factor of 86 and 68, respectively, from methanol-grown Methanosarcina barkeri cells. The dehydrogenase was isolated as a hexamer of a single 35 kDa subunit ... >> More

  5,10-Methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase have been purified to homogeneity by a factor of 86 and 68, respectively, from methanol-grown Methanosarcina barkeri cells. The dehydrogenase was isolated as a hexamer of a single 35 kDa subunit, whereas the reductase was composed of four identical 38 kDa subunits. The purified oxygen-stable enzymes catalyzed the oxidation of 5,10-methylenetetrahydromethanopterin and methyltetrahydromethanopterin with Vmax values of 3000 and 200 mumol min-1 mg-1, respectively. The methanogenic electron carrier coenzyme F420 was a specific electron acceptor for both enzymes. Steady state kinetics for the two enzymes were in agreement with ternary complex (sequential) mechanisms. Methylene reductase and methylene dehydrogenase are proposed to function in the methanol oxidation step to CO2. << Less

  Biochim Biophys Acta 1079:293-302(1991) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and properties of N5, N10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg).

  Ma K., Thauer R.K.

  The reduction of N5,N10-methylenetrahydromethanopterin (CH2 = H4MPT) to N5-methyltetrahydromethanopterin (CH3-H4MPT) is an intermediate step in methanogenesis from CO2 and H2. The reaction is catalyzed by CH2 = H4MPT reductase. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) ... >> More

  The reduction of N5,N10-methylenetrahydromethanopterin (CH2 = H4MPT) to N5-methyltetrahydromethanopterin (CH3-H4MPT) is an intermediate step in methanogenesis from CO2 and H2. The reaction is catalyzed by CH2 = H4MPT reductase. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) was found to be specific for reduced coenzyme F420 as electron donor; neither NADH or NADPH nor reduced viologen dyes could substitute for the reduced 5-deazaflavin. The reductase was purified over 100-fold to apparent homogeneity. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band at the 36-kDa position. The apparent molecular mass of the native enzyme was determined by gel filtration to be in the order of 150 kDa. The purified enzyme was colourless. It did not contain flavin or iron. The ultraviolet visible spectrum was almost identical to that of albumin, suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentration at different constant concentrations of the second substrate yielded straight lines intersecting at one point on the abscissa to the left of the vertical axis. This intersecting pattern is characteristic of a ternary complex catalytic mechanism. The Km for CH2 = H4MPT and for the reduced coenzyme F420 were determined to be 0.3 mM and 3 microM, respectively. Vmax was 6000 mumol.min-1.mg protein-1 (kcat = 3600 s-1). The CH2 = H4MPT reductase was stable in the presence of air; at 4 C less than 10% activity was lost within 24 h. << Less

  Eur. J. Biochem. 191:187-193(1990) [PubMed] [EuropePMC]

• Purification and properties of N5,N10-methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) from the extreme thermophile Methanopyrus kandleri.

  Ma K., Linder D., Stetter K.O., Thauer R.K.

  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 methanogens known so far. N5,N10-Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogene ... >> More

  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 methanogens known so far. N5,N10-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. Vmax at 65 degrees C and pH 6.8 was 435 U/mg (kcat = 275 s-1) and the Km for methylenetetrahydromethanopterin and for reduced F420 were 6 microM and 4 microM, respectively. From Arrhenius plots an activation energy of 34 kJ/mol was determined. The Q10 between 40 degrees C and 90 degrees 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. << Less

  Arch. Microbiol. 155:593-600(1991) [PubMed] [EuropePMC]

• Purification and properties of 5,10-methylenetetrahydromethanopterin reductase, a coenzyme F420-dependent enzyme, from Methanobacterium thermoautotrophicum strain delta H.

  te Broemmelstroet B.W., Hensgens C.M.H., Keltjens J.T., van der Drift C., Vogels G.D.

  5,10-Methylenetetrahydromethanopterin reductase was purified 22-fold to apparent homogeneity from the methanogenic bacterium Methanobacterium thermoautotrophicum. The enzyme catalyzes the reduction of 5,10-methylene-to 5-methyltetrahydromethanopterin. The electron carrier coenzyme F420 is specific ... >> More

  5,10-Methylenetetrahydromethanopterin reductase was purified 22-fold to apparent homogeneity from the methanogenic bacterium Methanobacterium thermoautotrophicum. The enzyme catalyzes the reduction of 5,10-methylene-to 5-methyltetrahydromethanopterin. The electron carrier coenzyme F420 is specifically used as the cosubstrate. The reductase reaction may proceed in both directions, methylene reduction is, however, thermodynamically favored. In addition, the velocity of the reaction in this direction exceeds the reverse reaction by a factor of 26. The reductase is composed of a single subunit with an estimated Mr = 35,000. The active enzyme does not contain a flavin prosthetic group or iron-sulfur clusters, in contrast to 5,10-methylenetetrahydrofolate reductases purified from eukaryotic and eubacterial sources, which catalyze an analogous reaction as the methanogenic reductase. << Less

  J. Biol. Chem. 265:1852-1857(1990) [PubMed] [EuropePMC]