Aminoacyl-tRNA Specificity of a Ligase Catalyzing Non-ribosomal Peptide Extension

Peptide aminoacyl-transfer ribonucleic acid ligases (PEARLs) are amide-bond-forming enzymes that extend the main chain of peptides by using aminoacyl-tRNA (aa-tRNA) as a substrate. In this study, we investigated the substrate specificity of the PEARL BhaB_C^Ala from <i>Bacillus halodurans</i>, which utilizes Ala-tRNA^Ala. By leveraging flexizyme, a ribozyme capable of charging diverse acids onto a desired tRNA, we generated an array of aa-tRNAs in which we varied both the amino acid and the tRNA to dissect the substrate scope of BhaB_C^Ala. We demonstrate that BhaB_C^Ala catalyzes peptide extension with noncognate proteinogenic and noncanonical amino acids, hydroxy acids, and mercaptocarboxylic acids when attached to tRNA^Ala. For most of these, the efficiency was considerably reduced compared to Ala, indicating that the enzyme recognizes the amino acid. By variation of the different parts of the tRNA, enzyme specificity was shown to also depend on the acceptor stem and the anticodon arm of the tRNA. These findings establish the molecular determinants of PEARL specificity and provide a foundation for engineering these enzymes for broader applications in peptide synthesis.