N5,N10-Methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum (strain Marburg) was purified under anaerobic conditions to apparent homogeneity and a specific activity of approximately 750 μol/min/mg protein. Polyacrylamide gel electrophoresis under native and denaturing conditions revealed that the enzyme is composed of only one polypeptide with an apparent molecular mass of 43 kDa. The purified enzyme catalyzed the dehydrogenation of N5,N10-methylenetetrahydromethanopterin (CH2=H4MPT) (apparent Km≡20 μM) to N5,N10-methenyltetrahydromethanopterin (CH≡H4MPT) in the absence of any added electron acceptors. One mol of H2 was generated per mol CH≡H4MPT formed, indicating that protons served as electron acceptor. Coenzyme F420, NAD, NADP and viologen dyes were not reduced by CH2=H4MPT. The dehydrogenase also catalyzed the reverse reaction, the reduction of CH≡H4MPT to CH2=H4MPT with H2. The data indicate that CH2=H4MPT dehydrogenase from M. thermoautotrophicum is a novel type of hydrogenase.
