Angew. Chem. Int. Ed. Engl. 10.1002/anie.201410047

The enzymatic incorporation of non-natural amino acids into proteins currently relies on self-acylating 'flexizymes' and engineered tRNA synthetases with altered substrate specificity. However, engineering synthetases to accept new substrates of interest is not always straightforward, and flexizymes are limited to cell-free translation. As an alternative approach, Giessen et al. envisioned that the analogous chemistry performed by adenylation (A) domains found within nonribosomal peptide synthetases could be coopted for use as surrogate tRNA synthetases, allowing facile access to hundreds of amino acid substrates. To test their hypothesis, the authors designed a fusion protein with the tRNA-recruiting multisynthetase complex accessory protein Arc1p to direct PheA as a representative A domain to a non-native tRNA substrate. Modeling based on an existing PheA structure and a newly acquired crystal structure of Arc1p suggested that a short linker connecting Arc1p to the C terminus of PheA would facilitate catalysis. Adenylation assays of constructs containing one of four linkers confirmed that PheA's activity was not disrupted by the Arc1p fusion, whereas aminoacylation assays using tRNAPhe further demonstrated that the fusion proteins were able to catalyze tRNA loading. The best construct, containing an 8-amino-acid linker, displayed ∼10% efficiency as compared to a native phenylalanine synthetase, PheRS; though this activity leaves room for improvement, it is likely sufficient for initial applications. Importantly, the reactions were equally efficient with L- or D-phenylalanine in the PheA constructs, whereas D-phenylalanine was only minimally processed by PheRS. As Arc1p is known to bind multiple tRNAs, the authors confirmed five other tRNAs could also be loaded with L- or D-phenylalanine. Analytical methods and a functional assay using a cyclodipeptide synthase, which uses amino acid–loaded tRNAs as substrates, further confirmed amino acid attachment at the expected terminal adenosine. Though future work will be needed to engender specificity in tRNA interactions if this method is to be used in cells, the approach opens up new opportunities for amino acid incorporation based on A domain diversity.