Schematic of pyrrolysine and methylamine metabolism in Methanosarcina spp. Pyrrolysine is made from two molecules of lysine. In the proposed pathway, PylB converts one lysine into a methylated D-ornithine derivative, which is then ligated to another lysine by PylC. The resultant dipeptide is oxidized by PylD, which results in spontaneous elimination of water and formation of pyrrolysine. Pyrrolysine is then ligated to tRNA^Pyl by PylS. The pyrrolysyl-tRNA^Pyl is carried to the ribosome by the usual elongation factor for co-translational incorporation into one of the three methylamine methyltransferases, MtmB, MtbB, or MttB. Below each methyltransferase is indicated its particular methylamine substrate. Pyrrolysine in the catalytic site is hypothesized to form an adduct with that methylamine which orients and activates it for methyl group transfer to the Co(I) form of the corrinoid cofactor bound to either MtmC, MtbB, or MttB. Each corrinoid protein interacts preferentially with the methyltransferase indicated below it. Adventitious oxidation of the corrinoid protein can result in inactivation, and a single protein, RamA, can return the corrinoid protein to the Co(I) state. All three methylated corrinoid proteins can serve as substrates for MtbA, which methylates the thiol of CoM (HSCoM). Methyl-CoM can then serve to directly generate methane, or to enter pathways leading to carbon assimilation and carbon dioxide production. Further details are provided in the text.

Local context of homologs of pyl and amber methylamine methyltransferase genes identified in completely sequenced genomes curated at the National Center for Biotechnology (NCBI). Colors indicate known functions of the homologous gene in Methanosarcina spp.: blue, pyrrolysine biosynthesis; red, amber decoding as pyrrolysine; green, methylamine metabolism; and gray, function unknown or not known to be involved in pyrrolysine or methylamine metabolism in Methanosarcina spp. All organisms with a full set of the pyl genes also have at least one mttB, mtbB, mtmB gene with the conserved amber codon (TAG), not all amber-methyltransferase genes are shown for each species. Numbers below genes indicate nucleotide location in genome. The Bilophila wadworthia pylT was identified on contig 1.181 (NZ_ADCP01000181.1), and the genome numbering is from the wgs scaffold at NCBI.

Secondary structure of tRNA^Pyl from Methanosarcina acetivorans and Desulfitobacterium hafniense. A). The tRNA^Pyl common to M. acetivorans, M. barkeri Fusaro and M. mazei is shown. Arrows indicate base substitutions found in M. burtonii (upper case), M. mahii (lower case), and M. evestigatum (lower case italics). Asterisks indicate positions at which base substitution resulted in less than 50% of UAG codon translation in a recombinant system [38]. B). The Desulfitobacterium hafniense tRNA^Pyl secondary structure. Colored residues indicate: dark orange, identical in archaeal and bacterial tRNA^Pyl; light orange, identical in bacterial tRNA^Pyl; and green, conserved (>70% identity) in bacterial tRNA^Pyl. Asterisks indicate bases contacting protein residues in D. hafniense PylSc:tRNA co-crystal [35].