title: Structural Elucidation of the Bispecificity of A Domains as a Basis for Activating Non-natural Amino Acids.
abstract: Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non-ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered A domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A1) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the A domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created A domains that were monospecific, or changed the substrate specificity to tryptophan. The non-natural amino acid 4-azidophenylalanine is also efficiently activated by a mutant A domain, thus enabling the production of diversified non-ribosomal peptides for bioorthogonal labeling.
authors.affiliation: European Molecular Biology Laboratory Hamburg Outstation c/o DESY, Notkestrasse 85, 22603 Hamburg (Germany).
authors.forename: Heidi
authors.initials: H
authors.lastname: Kaljunen
authors.affiliation: University of Muenster, Department of Chemistry, Institute of Biochemistry, Wilhelm-Klemm-Straße 2, 48149 Muenster (Germany).
authors.forename: Stephan H H
authors.initials: SH
authors.lastname: Schiefelbein
authors.affiliation: University of Muenster, Department of Chemistry, Institute of Biochemistry, Wilhelm-Klemm-Straße 2, 48149 Muenster (Germany).|Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Münster (Germany).
authors.forename: Daniela
authors.initials: D
authors.lastname: Stummer
authors.affiliation: European Molecular Biology Laboratory Hamburg Outstation c/o DESY, Notkestrasse 85, 22603 Hamburg (Germany).
authors.forename: Sandra
authors.initials: S
authors.lastname: Kozak
authors.affiliation: European Molecular Biology Laboratory Hamburg Outstation c/o DESY, Notkestrasse 85, 22603 Hamburg (Germany).
authors.forename: Rob
authors.initials: R
authors.lastname: Meijers
authors.affiliation: University of Innsbruck, Research Institute for Limnology, Mondseestr. 9, 5310 Mondsee (Austria). guntram.christiansen@uibk.ac.at.
authors.forename: Guntram
authors.initials: G
authors.lastname: Christiansen
authors.affiliation: University of Muenster, Department of Chemistry, Institute of Biochemistry, Wilhelm-Klemm-Straße 2, 48149 Muenster (Germany). a.rentmeister@uni-muenster.de.|Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Münster (Germany). a.rentmeister@uni-muenster.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Rentmeister
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D054300:Oscillatoria / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D001386:Azides; D010456:Peptides, Cyclic; C027162:4-azidophenylalanine; D010649:Phenylalanine; D010453:Peptide Synthases
keywords: adenylation domains; crystal structures; cyanobacteria; non-ribosomal peptide synthetases; substrate specificity