Substrate recognition by a peptide- aminoacyl- tRNA ligase

• 成果类型: Article
• 署名作者: Ramos-Figueroa, Josseline; Liang, Haoqian; van der Donk, Wilfred A.
• 署名单位: University of Illinois System; University of Illinois Urbana-Champaign; University of Illinois System; University of Illinois Urbana-Champaign; Howard Hughes Medical Institute; University of Illinois System; University of Illinois Urbana-Champaign
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-12618
• DOI: 10.1073/pnas.2423858122
• 发表日期: 2024-03-25
• 关键词: genetic-code; biosynthesis; acids; expression; proteins; insights; scaffold

摘要

The continuing discovery of new peptide-aminoacyl-tRNA ligases (PEARLs) has unveiled a diverse array of enzymes with the unique potential to append amino acids to the C terminus of substrate peptides in an aminoacyl-tRNA-dependent manner. To date, PEARLs have been reported that can conjugate Cys, Ala, Trp, Gly, Leu, Asn, and Thr residues, but the basis of peptide substrate and aminoacyl-tRNA recognition is not known. Cell-free expression (CFE) has emerged as a powerful tool to rapidly assay activity of substrate variants, and we used the technique in this study to investigate the peptide substrate specificity of the PEARL BhaBTrp C . This enzyme that adds Trp was discovered previously during genome mining for ribosomally synthesized and post-translational modified peptides (RiPPs). The enzyme is remarkably tolerant of changes to the C-terminal amino acid of the peptide substrate, and truncation and replacement experiments suggest a minimal sequence requirement. An AlphaFold3 model provided insights into binding interactions of the substrate peptide BhaA-Ala to BhaBTrp C and also generated predictions for tRNA, ATP, and Mg2+ binding modes that were tested by site-directed mutagenesis. The data suggest that several highly conserved residues in PEARLs recognize the 3 '-CCA sequence present in all tRNAs. The minimal sequence required for Trp incorporation by BhaBTrp C was employed as a protein tag for C-terminal labeling of eGFP, lysozyme, and MBP with Trp and 5-Br-Trp.