COENZYME F-420-DEPENDENT N-5,N-10-METHYLENETETRAHYDROMETHANOPTERIN REDUCTASE (MER) FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM STRAIN MARBURG CLONING, SEQUENCING, TRANSCRIPTIONAL ANALYSIS, AND FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI OF THE MER GENE

The gene encoding the F-420-dependent N-5,N-10-methylenetetrahydromethanopterin reductase (Mer), which catalyzes an intermediate step in methanogensis, was cloned and sequenced from the thermophilic Methanobacterium thermoautotrophicum strain Marburg. The gene was identified on a 3.8-kbp BamHI fragment of M. thermoautotrophicum genomic DNA using a homologous probe. The mer gene encoded an acidic protein of 321 amino acids, corresponding to a calculated molecular mass of 33492 Da. Sequence analysis revealed the presence of a ribosome binding site, a putative promoter, and a possible terminator structure. The size of the mer mRNA was estimated as 1 kb indicating monocistronic transcription. The mer gene was expressed in Escherichia coli yielding an active enzyme of 36 kDa consistent with the apparent molecular mass described for the enzyme from M. thermoautotrophicum. Sequence comparisons revealed similarities between the F-420-dependent N-5,N-10-methylenetetrahydromethanopterin reductase and a F-420-dependent reductase involved in lincomycin biosynthesis in Streptomyces lincolnensis.