Publications

• Crystal structure of methylornithine synthase (PylB): insights into the pyrrolysine biosynthesis.

  Quitterer F., List A., Eisenreich W., Bacher A., Groll M.

  Angew. Chem. Int. Ed. 51:1339-1342(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Biosynthesis of the 22nd genetically encoded amino acid pyrrolysine: structure and reaction mechanism of PylC at 1.5A resolution.

  Quitterer F., List A., Beck P., Bacher A., Groll M.

  The second step in the biosynthesis of the 22nd genetically encoded amino acid pyrrolysine (Pyl) is catalyzed by PylC that forms the pseudopeptide L-lysine-N(ε)-3R-methyl-D-ornithine. Here, we present six crystal structures of the monomeric active ligase in complex with substrates, reaction interm ... >> More

  The second step in the biosynthesis of the 22nd genetically encoded amino acid pyrrolysine (Pyl) is catalyzed by PylC that forms the pseudopeptide L-lysine-N(ε)-3R-methyl-D-ornithine. Here, we present six crystal structures of the monomeric active ligase in complex with substrates, reaction intermediates, and products including ATP, the non-hydrolyzable ATP analogue 5'-adenylyl-β-γ-imidodiphosphate, ADP, D-ornithine (D-Orn), L-lysine (Lys), phosphorylated D-Orn, L-lysine-N(ε)-D-ornithine, inorganic phosphate, carbonate, and Mg(2+). The overall structure of PylC reveals similarities to the superfamily of ATP-grasp enzymes; however, there exist unique structural and functional features for a topological control of successive substrate entry and product release. Furthermore, the presented high-resolution structures provide detailed insights into the reaction mechanism of isopeptide bond formation starting with phosphorylation of D-Orn by transfer of a phosphate moiety from activated ATP. The binding of Lys to the enzyme complex is then followed by an S(N)2 reaction resulting in L-lysine-N(ε)-D-ornithine and inorganic phosphate. Surprisingly, PylC harbors two adenine nucleotides bound at the active site, what has not been observed in any ATP-grasp protein analyzed to date. Whereas one ATP molecule is involved in catalysis, the second adenine nucleotide functions as a selective anchor for the C- and N-terminus of the Lys substrate and is responsible for protein stability as shown by mutagenesis. << Less

  J. Mol. Biol. 424:270-282(2012) [PubMed] [EuropePMC]

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

  Gaston M.A., Zhang L., Green-Church K.B., Krzycki J.A.

  Pyrrolysine, the twenty-second amino acid found to be encoded in the natural genetic code, is necessary for all of the known pathways by which methane is formed from methylamines. Pyrrolysine comprises a methylated pyrroline carboxylate in amide linkage to the ε-amino group of L-lysine. In certain ... >> More

  Pyrrolysine, the twenty-second amino acid found to be encoded in the natural genetic code, is necessary for all of the known pathways by which methane is formed from methylamines. Pyrrolysine comprises a methylated pyrroline carboxylate in amide linkage to the ε-amino group of L-lysine. In certain Archaea, three methyltransferases initiate methanogenesis from the various methylamines, and these enzymes are encoded by genes with an in-frame amber codon that is translated as pyrrolysine. Escherichia coli that has been transformed with the pylTSBCD genes from methanogenic Archaea can incorporate endogenously biosynthesized pyrrolysine into proteins. The decoding of UAG as pyrrolysine requires pylT, which produces tRNA(Pyl) (also called tRNA(CUA)), and pylS, which encodes a pyrrolysyl-tRNA synthetase. The pylB, pylC and pylD genes are each required for tRNA-independent pyrrolysine synthesis. 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. << Less

  Nature 471:647-650(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• D-Ornithine coopts pyrrolysine biosynthesis to make and insert pyrroline-carboxy-lysine.

  Cellitti S.E., Ou W., Chiu H.P., Gruenewald J., Jones D.H., Hao X., Fan Q., Quinn L.L., Ng K., Anfora A.T., Lesley S.A., Uno T., Brock A., Geierstanger B.H.

  D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and b ... >> More

  D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl. << Less

  Nat. Chem. Biol. 7:528-530(2011) [PubMed] [EuropePMC]