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 BhaBCAla from Bacillus halodurans, which utilizes
Ala-tRNAAla. 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 BhaBCAla. We
demonstrate that BhaBCAla catalyzes peptide
extension with noncognate proteinogenic and noncanonical amino acids,
hydroxy acids, and mercaptocarboxylic acids when attached to tRNAAla. 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.