The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine

Pyrrolysine, the latest addition to the ranks of the genetically encoded amino acids, is found in methanogenic archaea and bacteria, in which it is a necessary component of the enzymes involved in the formation of methane from methylamines. Its biosynthetic pathway remains poorly characterized. Joseph Krzycki and colleagues now show that lysine is the sole precursor for the pyrrolysine synthesis system of Methanosarcina sp. Archaea. The radical S-adenosyl-L-methionine protein PylB converts lysine to 3-methylornithine, which then undergoes a PylC-catalysed condensation with a second lysine to generate a product that is oxidized by PylD to generate pyrrolysine. The authors suggest that synthetic analogues of intermediates in this pathway may provide useful pyrrolysine derivatives that could be incorporated into tailored recombinant proteins. Pyrrolysine is the twenty-second naturally occurring amino acid, and it is required for the formation of methane from methylamines. Pyrrolysine is the last remaining natural amino acid with a poorly characterized biosynthetic pathway. Here it is shown that the radical SAM protein PylB converts lysine to 3-methylornithine, which then undergoes a PylC-catalysed condensation with another lysine to generate a product that is oxidized by PylD to generate pyrrolysine. Pyrrolysine, the twenty-second amino acid found to be encoded in the natural genetic code1,2,3,4, is necessary for all of the known pathways by which methane is formed from methylamines5,6. Pyrrolysine comprises a methylated pyrroline carboxylate in amide linkage to the e-amino group of l-lysine2,7,8. In certain Archaea, three methyltransferases initiate methanogenesis from the various methylamines9,10,11, and these enzymes are encoded by genes with an in-frame amber codon12,13 that is translated as pyrrolysine2,7,8. Escherichia coli that has been transformed with the pylTSBCD genes from methanogenic Archaea can incorporate endogenously biosynthesized pyrrolysine into proteins14. The decoding of UAG as pyrrolysine requires pylT1,6, which produces tRNAPyl (also called tRNACUA), and pylS1, which encodes a pyrrolysyl-tRNA synthetase4,15,16. The pylB, pylC and pylD genes1 are each required for tRNA-independent pyrrolysine synthesis14. Pyrrolysine is the last remaining genetically encoded amino acid with an unknown biosynthetic pathway. Here we provide genetic and mass spectrometric evidence for a pylBCD-dependent pathway in which pyrrolysine arises from two lysines. We show that a newly uncovered UAG-encoded amino acid, desmethylpyrrolysine, is made from lysine and exogenous d-ornithine in a pylC-dependent process followed by a pylD-dependent process, but it is not further converted to pyrrolysine. These results indicate that the radical S-adenosyl-l-methionine (SAM) protein PylB mediates a lysine mutase reaction that produces 3-methylornithine, which is then ligated to a second molecule of lysine by PylC before oxidation by PylD results in pyrrolysine. The discovery of lysine as the sole precursor to pyrrolysine will further inform discussions of the evolution of the genetic code and amino acid biosynthetic pathways. Furthermore, intermediates of the pathway may provide new avenues by which the pyl system can be exploited to produce recombinant proteins with useful modified residues.