A Synthetic Adenylation‐Domain‐Based tRNA‐Aminoacylation Catalyst |
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| Authors: | Dr Tobias W Giessen Florian Altegoer Annika J Nebel Roman M Steinbach Dr Gert Bange Prof?Dr Mohamed A Marahiel |
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| Affiliation: | 1. Department of Chemistry, Philipps‐University Marburg, Hans‐Meerwein‐Strasse 4, 35032 Marburg (Germany);2. LOEWE Center for Synthetic Microbiology (Synmikro), Philipps‐University Marburg, Hans‐Meerwein‐Strasse, 35032 Marburg (Germany) |
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| 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. |
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| Keywords: | biocatalysis peptides protein engineering protein structure RNA |
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