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

J Mol Biol. 2012 Dec 14;424(5):270-82. doi: 10.1016/j.jmb.2012.09.007. Epub 2012 Sep 14.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biosynthetic Pathways*
  • Catalytic Domain
  • Crystallography, X-Ray
  • Lysine / analogs & derivatives*
  • Lysine / biosynthesis
  • Methanosarcina barkeri / enzymology*
  • Models, Molecular
  • Molecular Sequence Data
  • Phosphotransferases / chemistry*
  • Protein Conformation

Substances

  • Phosphotransferases
  • pyrrolysine
  • Lysine

Associated data

  • PDB/4FFL
  • PDB/4FFM
  • PDB/4FFN
  • PDB/4FFO
  • PDB/4FFP
  • PDB/4FFR