A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry
| Autor: | Anna Joëlle Ruff, Alexandra Maria Weingartner, Patrik Fladischer, Birgit Wiltschi, Ulrich Schwaneberg, Johannes Blamauer, Ruth Birner-Gruenberger, Tsvetan Kardashliev, Barbara Darnhofer |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Azides Desulfitobacterium medicine.disease_cause 01 natural sciences Applied Microbiology and Biotechnology Substrate Specificity Amino Acyl-tRNA Synthetases 03 medical and health sciences chemistry.chemical_compound Phenyl azide medicine Escherichia coli Amino Acids chemistry.chemical_classification biology Methanosarcina mazei 010405 organic chemistry Methanocaldococcus jannaschii Desulfitobacterium hafniense General Medicine biology.organism_classification 0104 chemical sciences Amino acid 030104 developmental biology chemistry Biochemistry Transfer RNA Methanosarcina Molecular Medicine Bacteria |
| Zdroj: | Biotechnology journal. 14(3) |
| ISSN: | 1860-7314 |
| Popis: | The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNACUA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNACUA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity. |
| Databáze: | OpenAIRE |
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