Two methylenetetrahydromethanopterin dehydrogenases have been purified from Methanobacterium thermoautotrophicum strain Marburg: one (MTD) is coenzyme F-dependent and oxygen-stable (Mukhopadhyay, B., and Daniels, L.(1989) Can. J. Microbiol. 35, 499-507), and the other (MTH) is coenzyme F-independent (or hydrogenase-type) and oxygen-sensitive (Zirngibl, C., Hedderich, R., and Thauer, R. K.(1990) FEBS Lett. 261, 112-116). Based on the NH-terminal sequence of MTD, a 36-mer oligonucleotide was designed and used to identify and clone a 6.1-kilobase pair EcoRI fragment of M. thermoautotrophicum DNA. Sequencing of this fragment revealed an 825-base pair (bp) MTD encoding gene (mtd), which was expressed in Escherichia coli yielding an enzyme that, like the native enzyme, was oxygen-stable, strictly dependent on coenzyme F, thermostable, thermophilic, and exhibited maximum activity at an acidic pH. The amino acid sequence predicts that MTD is a hydrophobic and acidic protein with no identifiable homology to MTH (von Bunau, R., Zirngibl, C., Thauer, R. K., and Klein, A.(1991) Eur. J. Biochem. 202, 1205-1208), but comparisons with coenzyme F utilizing enzymes revealed a conserved region at the NH terminus of MTD that could correspond to the ability to interact with coenzyme F. The mtd transcript was 900 nucleotides long and initiated 8 bp upstream of the translation initiation codon and 22 bp downstream from an archaeal promoter sequence. The mtd coding sequence was followed by several poly(dT) sequences and an inverted repeat that could be transcription termination signals.

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