Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases.
basic_science · Level V
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- Record sourced from PubMed, PMID 42045211.
- Also identified by DOI 10.1038/s41467-026-72367-y.
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Abstract
Peptide asparaginyl ligases (PALs) are powerful tools for protein engineering but are limited by natural rarity and poor expression. We mine 23 cyclotide-rich Viola species, uncovering 29 PALs that expand the known repertoire to 47. A dual-objective screen identifies VdiPAL1 as the best-performed natural PAL, with twice efficiency of wt-VyPAL2 and 12 mg L<sup>-1</sup> soluble expression in E. coli. A broad P2" specificity including Trp/Ile/Leu/Phe/Tyr/Met is discovered across diverse PALs, which enables sequential click-compatible liposome dual-functionalization. 1.8 Å 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 yields 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.