Purification and properties of N5, N10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg).
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.
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Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Federal Republic of Germany.
Thauer
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RK
D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D019605:Euryarchaeota / Q000201:enzymology*; D005415:Flavins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification*; D011489:Protein Denaturation; D013696:Temperature
D005415:Flavins; D007501:Iron; D000587:Oxidoreductases Acting on CH-NH Group Donors; C047126:methylenetetrahydromethanopterin dehydrogenase