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-1 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.
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Acknowledgements
We thank the botanist Ce-hong Li (Emei Mountain Biotic Resource Experimental Station of China) for aiding on field collection of Viola plants, Julien Lescar (Nanyang Institute of Structural Biology, Singapore) for advice on crystallography data analysis, and the staff of beamline BL02U1 at the Shanghai Synchrotron Radiation Facility for assistance during the data collection. This work was supported by the National Key Research and Development Program of China (2023YFA0916000, X.H.), National Natural Science Foundation of China (32371324, X.H.), the Key R&D Program of Hubei Jiangxia Laboratory (JXBS010, G-W.H.), and the National Natural Science Foundation of China (32270228, G-W.H.).
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Du, W., Qi, S., Zhen, M. et al. Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases. Nat Commun (2026). https://doi.org/10.1038/s41467-026-72367-y
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DOI: https://doi.org/10.1038/s41467-026-72367-y
