Purification and properties of N 5 , N 10 ‐methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg)

The reduction of N 5 ,N 10 ‐methylenetetrahydromethanopterin (CH 2 = H 4 MPT) to N 5 ‐methyltetrahydromethanopterin (CH 3 ‐H 4 MPT) is an intermediate step in methanogenesis from CO 2 and H 2 . The reaction is catalyzed by CH 2 = H 4 MPT reductase. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) was found to be specific for reduced coenzyme F 420 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 K m for CH 2 = H 4 MPT and for the reduced coenzyme F 420 were determined to be 0.3 mM and 3 μM, respectively. V max was 6000 μmol · min −1 · mg protein −1 ( K cat = 3600 s −1 ). The CH 2 = H 4 MPT reductase was stable in the presence of air; at 4°C less than 10% activity was lost within 24 h.