Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-l-a-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept a-hydroxy, a-thio and N-formyl-l-a-amino acids, as well as a-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an a-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose a-substituent diverges substantially from the a-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

Automated summary

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-l-a-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants have been found to accept a-hydroxy, a-thio and N-formyl-l-a-amino acids, as well as a-carboxy acid monomers that are precursors to polyketide natural products. This discovery highlights the potential of PylRS-derived enzymes in expanding the range of monomers used in protein synthesis.