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 -α-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 acc...
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Veröffentlicht in: | Nature chemistry 2023-07, Vol.15 (7), p.960-971 |
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Sprache: | eng |
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Zusammenfassung: | The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-
l
-α-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 α-hydroxy, α-thio and
N
-formyl-
l
-α-amino acids, as well as α-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 α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-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.
Ribosomal incorporation of non-α-amino acid monomers into proteins is largely restricted to in vitro translation. Now, pyrrolysyl-transfer RNA synthetase variants have been shown to acylate tRNAs with α-thio acids, malonic acids, and
N
-formyl amino acids. This work represents a key step towards the programmed ribosomal synthesis of sequence-defined non-protein polymers in cellulo. |
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ISSN: | 1755-4330 1755-4349 1755-4349 |
DOI: | 10.1038/s41557-023-01224-y |