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

Pyrrolysine, the 22(nd) amino acid to be found in the natural genetic code1–4, is necessary for all known pathways of methane formation from methylamines5,6. The residue is comprised of a methylated pyrroline carboxylate in amide linkage to the ε-amino group of L-lysine2,7,8. The three different methyltransferases that initiate methanogenesis from different methylamines9–11 have genes with an in-frame amber codon12,13 translated as pyrrolysine2,7,8. E. coli transformed with pylTSBCD from methanogenic Archaea can incorporate endogenously biosynthesized pyrrolysine into protein14. The decoding of UAG as pyrrolysine requires pylT1,6 which produces tRNA(Pyl) (also called tRNA(CUA)), and pylS1 encoding a pyrrolysyl-tRNA synthetase4,15,16. The pylBCD genes1 are each required for tRNA-independent pyrrolysine synthesis14. Pyrrolysine has been the last remaining genetically encoded amino acid with an unknown biosynthetic pathway. Here, we provide genetic and mass spectroscopic evidence for a pylBCD-dependent pathway in which pyrrolysine arises from two lysines. We show that a new UAG encoded residue, desmethylpyrrolysine, is made from lysine and exogenous D-ornithine in a pylC, then a pylD, dependent process, but is not further converted to pyrrolysine. These results indicate that the radical S-adenosyl-methionine (SAM) protein PylB mediates a lysine mutase reaction producing 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 sole precursor to pyrrolysine will further inform discussions of the evolution the genetic code and amino acid biosynthetic pathways, while intermediates of the pathway may provide new avenues by which the pyl system may be exploited for production of recombinant proteins with useful modified residues.