Biosynthesis of the 22nd Genetically Encoded Amino Acid Pyrrolysine: Structure and Reaction Mechanism of PylC at 1.5Å Resolution
Type
ArticleKAUST Grant Number
FIC/2010/07Date
2012-12Permanent link to this record
http://hdl.handle.net/10754/597678
Metadata
Show full item recordAbstract
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.Citation
Quitterer F, List A, Beck P, Bacher A, Groll M (2012) Biosynthesis of the 22nd Genetically Encoded Amino Acid Pyrrolysine: Structure and Reaction Mechanism of PylC at 1.5Å Resolution. Journal of Molecular Biology 424: 270–282. Available: http://dx.doi.org/10.1016/j.jmb.2012.09.007.Sponsors
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.Publisher
Elsevier BVJournal
Journal of Molecular BiologyPubMed ID
22985965ae974a485f413a2113503eed53cd6c53
10.1016/j.jmb.2012.09.007
Scopus Count
Collections
Publications Acknowledging KAUST SupportRelated articles
- Reactivity and chemical synthesis of L-pyrrolysine- the 22(nd) genetically encoded amino acid.
- Authors: Hao B, Zhao G, Kang PT, Soares JA, Ferguson TK, Gallucci J, Krzycki JA, Chan MK
- Issue date: 2004 Sep
- The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine.
- Authors: Gaston MA, Zhang L, Green-Church KB, Krzycki JA
- Issue date: 2011 Mar 31
- Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase.
- Authors: Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S
- Issue date: 2008 May 2
- Crystal structure of methylornithine synthase (PylB): insights into the pyrrolysine biosynthesis.
- Authors: Quitterer F, List A, Eisenreich W, Bacher A, Groll M
- Issue date: 2012 Feb 6
- Crystal structures of catalytic intermediates of human selenophosphate synthetase 1.
- Authors: Wang KT, Wang J, Li LF, Su XD
- Issue date: 2009 Jul 24