Biosynthesis of the 22nd Genetically Encoded Amino Acid Pyrrolysine: Structure and Reaction Mechanism of PylC at 1.5Å Resolution

Handle URI:
http://hdl.handle.net/10754/597678
Title:
Biosynthesis of the 22nd Genetically Encoded Amino Acid Pyrrolysine: Structure and Reaction Mechanism of PylC at 1.5Å Resolution
Authors:
Quitterer, Felix; List, Anja; Beck, Philipp; Bacher, Adelbert; Groll, Michael
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-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.
Publisher:
Elsevier BV
Journal:
Journal of Molecular Biology
KAUST Grant Number:
FIC/2010/07
Issue Date:
Dec-2012
DOI:
10.1016/j.jmb.2012.09.007
PubMed ID:
22985965
Type:
Article
ISSN:
0022-2836
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.
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DC FieldValue Language
dc.contributor.authorQuitterer, Felixen
dc.contributor.authorList, Anjaen
dc.contributor.authorBeck, Philippen
dc.contributor.authorBacher, Adelberten
dc.contributor.authorGroll, Michaelen
dc.date.accessioned2016-02-25T12:44:15Zen
dc.date.available2016-02-25T12:44:15Zen
dc.date.issued2012-12en
dc.identifier.citationQuitterer 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.en
dc.identifier.issn0022-2836en
dc.identifier.pmid22985965en
dc.identifier.doi10.1016/j.jmb.2012.09.007en
dc.identifier.urihttp://hdl.handle.net/10754/597678en
dc.description.abstractThe 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.en
dc.description.sponsorshipWe 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.en
dc.publisherElsevier BVen
dc.subjectamino aciden
dc.subjectATP-grasp enzymeen
dc.subjectbiosynthesis of pyrrolysineen
dc.subjectisopeptide bond formationen
dc.subjectX-ray crystallographyen
dc.titleBiosynthesis of the 22nd Genetically Encoded Amino Acid Pyrrolysine: Structure and Reaction Mechanism of PylC at 1.5Å Resolutionen
dc.typeArticleen
dc.identifier.journalJournal of Molecular Biologyen
dc.contributor.institutionTechnische Universitat Munchen, Munich, Germanyen
kaust.grant.numberFIC/2010/07en

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