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

The gene encoding the F-420-dependent N-5,N-10-methylenetetrahydrometh anopterin reductase (Mer), which catalyzes an intermediate step in met hanogensis, was cloned and sequenced from the thermophilic Methanobact erium 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. Seque nce analysis revealed the presence of a ribosome binding site, a putat ive promoter, and a possible terminator structure. The size of the mer mRNA was estimated as 1 kb indicating monocistronic transcription. Th e mer gene was expressed in Escherichia coli yielding an active enzyme of 36 kDa consistent with the apparent molecular mass described for t he enzyme from M. thermoautotrophicum. Sequence comparisons revealed s imilarities between the F-420-dependent N-5,N-10-methylenetetrahydrome thanopterin reductase and a F-420-dependent reductase involved in linc omycin biosynthesis in Streptomyces lincolnensis.