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-Nε-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-Nε-d-ornithine, inorganic phosphate, carbonate, and Mg2 +. 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 SN2 reaction resulting in l-lysine-Nε-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. © 2012 Elsevier Ltd.
Bibliographical noteKAUST Repository Item: Exported on 2020-10-01
Acknowledged KAUST grant number(s): FIC/2010/07
Acknowledgements: We thank the staff of the beamline X06SA at the Paul Scherer Institute, Swiss Light Source, Villigen, Switzerland, for their help with data collection and Katrin Gartner for excellent technical assistance. In addition, we thank our student Stephanie Heinzlmeir for her help. This work was supported by the Hans-Fischer Gesellschaft, Award No. FIC/2010/07 from the King Abdullah University of Science and Technology. (KAUST) and by the Deutsche Forschungsgemeinschaft (DFG), grant GR1861/7-1.
This publication acknowledges KAUST support, but has no KAUST affiliated authors.