A synthetic adenylation-domain-based tRNA-aminoacylation catalyst.

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.