title: A synthetic adenylation-domain-based tRNA-aminoacylation catalyst.
abstract: The incorporation of non-proteinogenic amino acids represents a major challenge for the creation of functionalized proteins. The ribosomal pathway is limited to the 20-22 proteinogenic amino acids while nonribosomal peptide synthetases (NRPSs) are able to select from hundreds of different monomers. Introduced herein is a fusion-protein-based design for synthetic tRNA-aminoacylation catalysts based on combining NRPS adenylation domains and a small eukaryotic tRNA-binding domain (Arc1p-C). Using rational design, guided by structural insights and molecular modeling, the adenylation domain PheA was fused with Arc1p-C using flexible linkers and achieved tRNA-aminoacylation with both proteinogenic and non-proteinogenic amino acids. The resulting aminoacyl-tRNAs were functionally validated and the catalysts showed broad substrate specificity towards the acceptor tRNA. Our strategy shows how functional tRNA-aminoacylation catalysts can be created for bridging the ribosomal and nonribosomal worlds. This opens up new avenues for the aminoacylation of tRNAs with functional non-proteinogenic amino acids.
authors.affiliation: Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany); LOEWE Center for Synthetic Microbiology (Synmikro), Philipps-University Marburg, Hans-Meerwein-Strasse, 35032 Marburg (Germany). tobias.giessen@chemie.uni-marburg.de.
authors.forename: Tobias W
authors.initials: TW
authors.lastname: Giessen
authors.forename: Florian
authors.initials: F
authors.lastname: Altegoer
authors.forename: Annika J
authors.initials: AJ
authors.lastname: Nebel
authors.forename: Roman M
authors.initials: RM
authors.lastname: Steinbach
authors.forename: Gert
authors.initials: G
authors.lastname: Bange
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D011302:Prephenate Dehydratase / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D046249:Transfer RNA Aminoacylation
chemical_list: D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C071216:P-protein, E coli; D011302:Prephenate Dehydratase; D000604:Amino Acyl-tRNA Synthetases
keywords: RNA; biocatalysis; peptides; protein engineering; protein structure