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Genes encoding methanogenic methylamine methyltransferases all contain an in-frame amber (UAG) codon that is read through during translation. We have identified the UAG-encoded residue in a 1.55 angstrom resolution structure of the Methanosarcina barkeri monomethylamine methyltransferase (MtmB). This structure reveals a homohexamer comprised of individual subunits with a TIM barrel fold. The electron density for the UAG-encoded residue is distinct from any of the 21 natural amino acids. Instead it appears consistent with a lysine in amide-linkage to (4R,SR)-4-substituted-pyrroline-5-carboxylate. We suggest that this amino acid be named L-pyrrolysine.
The catabolism of methylamines by methanogens involves a conserved arrangement of proteins. A specific monomethylamine (MMA), dimethylamine (DMA), or trimethylamine (TMA) methyltransferase activates the substrate for methyl transfer to a cognate corrinoid protein (1, 2). A second methyl-- transferase catalyzes the transfer of the methyl group from the methylated corrinoid cofactor to coenzyme M (CoM). Methyl-CoM is subsequently used to generate methane by methyl-CoM reductase (3).
All known methanogen methylamine (MMA, DMA, or TMA) methyltransferase genes contain a single in-frame amber (UAG) codon that does not appear to stop translation during protein synthesis (4, 5). This strict conservation contrasts with the lack of sequence similarity between different MMA, DMA, and TMA methyltransferase gene families. Analysis of tryptic fragments of MMA methyltransferase (MtmB) by mass spectrometry and Edman degradation suggested that the amber codon serves as a sense codon that corresponds to lysine (6). The harsh conditions of peptide isolation, however, left open the possibility that the amber codon signals a modified lysine residue. The use of what is normally a stop codon to signal the incorporation of an unusual amino acid has precedent in the use of UGA to encode selenocysteine, the 21st natural amino acid found in Bacterial, Eucaryal, and Archaeal proteins (7,8).
The structure of the Methanosarcina barkeri MS monomethylamine methyltransferase was determined to clarify the identity of the UAG-encoded residue (Tables 1 and 2). Two forms of the enzyme were obtained from crystallization conditions that differed only in the precipitating salt used [NaC1 for form 1, and (NH^sub 4^)^sub 2^SO^sub 4^ for form 2] and were solved to 1.55 Angstrom and 1.7 Angstrom resolution, respectively. The global conformations for these two forms are virtually identical, except for the region around the UAG-encoded amino...