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methanogenic Archaea, Methanosarcina barkeri, methyltransferases, corrinoids, uroporphyrinogen III
Abstract:
Methanosarcina barkeri is known to contain two methyltransferase isoenzymes, here designated MtaA and MtbA, which catalyze the formation of methyl-coenzyme M from methylcobalamin and coenzyme M. The genes encoding the two soluble 34-kDa proteins have been cloned and sequenced. mtaA and mtbA were found to be located in different parts of the genome, each forming a monocystronic transcription unit. Northern blot analysis revealed that mtaA is preferentially transcribed when M. barkeri is grown on methanol and the mtbA gene when the organism is grown on H2/CO2 or trimethylamine. Comparison of the deduced amino acid sequences revealed the sequences of the two isoenzymes to be 37% identical. Both isoenzymes showed sequence similarity to uroporphyrinogen III decarboxylase from Escherichia coli. The mtaA gene was tagged with a sequence encoding six His placed six bp before the mtaA start codon, and was functionally overexpressed in E. coli. 25% of the E. coli protein was found to be active methyltransferase which could be, purified in two steps to apparent homogenity with a 70% yield.
