Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases

Hemu, X.; Wenyu, D.; Qi, S.; Zhen, M.; Liao, M.; Zhao, Y.; Ma, J.; Hao, Y.; Jiang, H.; Lu, G.; Liew, C. W.; Chua, N.; Chen, H.; Hu, G.

doi:10.64898/2025.12.16.694575

bioRxiv · 10.64898/2025.12.16.694575

Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases

Hemu, X.·Wenyu, D.·Qi, S.·Zhen, M.·Liao, M.·Zhao, Y.·Ma, J.·Hao, Y.·Jiang, H.·Lu, G.·Liew, C. W.·Chua, N.·Chen, H.·Hu, G.

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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 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. 1.8L[A] 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 summaryMining 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.

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Hemu, X., Wenyu, D., Qi, S., Zhen, M., Liao, M., Zhao, Y., Ma, J., Hao, Y., Jiang, H., Lu, G., Liew, C. W., Chua, N., Chen, H., Hu, G.. 2025-12-16. Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases. https://doi.org/10.64898/2025.12.16.694575

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