Evolving the N‐Terminal Domain of Pyrrolysyl‐tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids

• Published in: Chembiochem : a European journal of chemical biology 2018-01, Vol.19 (1), p.26-30
• Main Authors: Sharma, Vangmayee, Zeng, Yu, Wang, W. Wesley, Qiao, Yuchen, Kurra, Yadagiri, Liu, Wenshe R.
• Format: Article
• Language: English
• Subjects: Amber; amber suppression; Amino acids; Amino Acyl-tRNA Synthetases - chemistry; Amino Acyl-tRNA Synthetases - genetics; Amino Acyl-tRNA Synthetases - metabolism; Biological evolution; Catalysis; Catalytic Domain; Efficiency; gene technology; Incorporation; Lysine; Lysine - analogs & derivatives; Lysine - metabolism; Methanosarcina - enzymology; Mutagenesis; Mutagenesis, Site-Directed; Mutants; Mutation; noncanonical amino acids; Protein Biosynthesis; Substrate Specificity; tRNA
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201700268

By evolving the N‐terminal domain of Methanosarcina mazei pyrrolysyl‐tRNA synthetase (PylRS) that directly interacts with tRNAPyl, a mutant clone displaying improved amber‐suppression efficiency for the genetic incorporation of Nϵ‐(tert‐butoxycarbonyl)‐l‐lysine threefold more than the wild type was identified. The identified mutations were R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of Nϵ‐acetyl‐l‐lysine and Nϵ‐(4‐azidobenzoxycarbonyl)‐l‐δ,ϵ‐dehydrolysine also improved the incorporation efficiency of these two noncanonical amino acids. As the three identified mutations were found in the N‐terminal domain of PylRS that was separated from its catalytic domain for charging tRNAPyl with a noncanonical amino acid, they could potentially be introduced to all other PylRS mutants to improve the incorporation efficiency of their corresponding noncanonical amino acids. Therefore, it represents a general strategy to optimize the pyrrolysine incorporation system‐based noncanonical amino‐acid mutagenesis. Watch the intake: By evolving the N‐terminal domain of pyrrolysyl‐tRNA synthetase (PylRS), mutations that improve the genetic incorporation of Nϵ‐(tertbutoxycarbonyl)‐l‐lysine at the amber codon are identified. These mutations can be directly transferred to PylRS mutants for improved incorporation efficiency of their corresponding noncanonical amino acids.