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

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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 ε-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.

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Figure 1: Proposed pathway of pyrrolysine biosynthesis from two molecules of lysine by the products of pylB, pylC and pylD.
Figure 2: UAG translation dependent on d -ornithine requires pylTSCD but not pylB.
Figure 3: Pathway of desmethylpyrrolysine biosynthesis.


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This work was supported by a grant from the National Institutes of Health to K.B.G.-C. and by grants from the US Department of Energy and the National Institutes of Health to J.A.K. The authors are grateful to D. Longstaff for preparing the tagged mtmB1 version of pDLBAD.

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M.A.G. planned experiments, labelled and purified MtmB samples from recombinant cells, made recombinant strains, carried out immunoblotting, analysed data and prepared figures. L.Z. carried out proteolytic digestion of MtmB, determined peptide and residue masses, analysed data and helped prepare data for presentation. K.B.G.-C. oversaw data acquisition and primary data analysis. J.A.K. planned and advised on experiments, analysed and interpreted data, prepared figures and wrote the manuscript.

Correspondence to Joseph A. Krzycki.

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Gaston, M., Zhang, L., Green-Church, K. et al. The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine. Nature 471, 647–650 (2011) doi:10.1038/nature09918

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