Work

Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases

Year: 2026

Type: article

Abstract: Peptide asparaginyl ligases (PALs) are powerful tools for protein engineering but are limited by natural rarity and poor expression. We mined 23 cyclotide-rich Viola species, uncovering 29 new PALs that expanded the known repertoire to 47. A dual-objective screen identified VdiPAL1 as the best-performed natural PAL, with twice efficiency of wt- VyPAL2 and 12 mg L -1 soluble expression in E. coli . A broad P2’’ specificity including Trp/Ile/Leu/Phe/Tyr/Met was discovered across diverse PALs, which enables sequential click-compatible liposome dual-functionalization. 1.8LÅ crystal structure of VdiPAL1 reveals a pre-organized near-attack conformation (NAC), supported by constant-pH MD simulations linking pH-dependent reactivity to NAC geometry. Our homology- and structure-based design yielded VyOpt1, a quintuple mutant of VyPAL2 with over 24-fold improved expression via enhanced cap-domain foldability in a single design-test cycle. This work expands the PAL family and demonstrates a transferable cap-domain-based engineering strategy, highlighting natural diversity as a powerful driver of enzyme discovery and optimization. TOC summary Mining of cyclotide-rich Viola genus expanded the total number of natural peptide asparaginyl ligases (PALs) to 47, including the discovery of highly expressible VdiPAL1. Its high-resolution structure provided new insights for mechanism, and its sequence guided us to a generalized PAL engineering strategy, leading to 24-fold increment in VyPAL2 expression.

Source: Nature Communications

DOI: 10.1038/s41467-026-72367-y