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Abstract 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 andN-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 am-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.
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Scalable and Selective β‐Hydroxy‐α‐Amino Acid Synthesis Catalyzed by Promiscuous l ‐Threonine Transaldolase ObiH
Abstract Enzymes from secondary metabolic pathways possess broad potential for the selective synthesis of complex bioactive molecules. However, the practical application of these enzymes for organic synthesis is dependent on the development of efficient, economical, operationally simple, and well‐characterized systems for preparative scale reactions. We sought to bridge this knowledge gap for the selective biocatalytic synthesis of β‐hydroxy‐α‐amino acids, which are important synthetic building blocks. To achieve this goal, we demonstrated the ability of ObiH, anl‐threonine transaldolase, to achieve selective milligram‐scale synthesis of a diverse array of non‐standard amino acids (nsAAs) using a scalable whole cell platform. We show how the initial selectivity of the catalyst is high and how the diastereomeric ratio of products decreases at high conversion due to product re‐entry into the catalytic cycle. ObiH‐catalyzed reactions with a variety of aromatic, aliphatic and heterocyclic aldehydes selectively generated a panel of β‐hydroxy‐α‐amino acids possessing broad functional‐group diversity. Furthermore, we demonstrated that ObiH‐generated β‐hydroxy‐α‐amino acids could be modified through additional transformations to access important motifs, such as β‐chloro‐α‐amino acids and substituted α‐keto acids.
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- Award ID(s):
- 1919350
- PAR ID:
- 10303566
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ChemBioChem
- Volume:
- 23
- Issue:
- 2
- ISSN:
- 1439-4227
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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