Skip to content
DraconiticResearch
Search Sources API
203,234 studies31 sourcesUpdated 47 min ago
Peer-reviewedCC BY-NC 3.0Chemical Science

Constrained diffusion-enabled construction of a highly expressed and robust peptide asparaginyl ligase

  • Shuo Pang1
  • Yuxing Hao2
  • Jiarong Mo1
  • Yingying He3
  • Yinglu Cui2
  • Bian Wu4
  • Xinya Hemu1
4 affiliations
  1. 1State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University Nanjing 211198 China
  2. 2State Key Laboratory of Microbial Diversity and Innovative Utilization, Institute of Microbiology, Chinese Academy of Sciences Beijing China
  3. 3School of Life Science and Technology, China Pharmaceutical University Nanjing 211198 China
  4. 4China State Key Laboratory of Green Biomanufacturing, Beijing University of Chemical Technology Beijing 100029 PR China
Read full textDOICite

Abstract

Peptide asparaginyl ligases (PALs) hold great promise for peptide macrocyclization and protein bioconjugation, yet their broader application is limited by insufficient thermal stability and low recombinant expression. Here, we report FortiPAL-1, a diffusion-enabled PAL developed through an integrated computational-experimental strategy that couples de novo sequence generation with multi-model-guided mutagenesis and PAL-specific functional refinements. Compared with natural PALs, FortiPAL-1 exhibits markedly enhanced thermal stability (Tm = 69 °C versus ∼50 °C for typical PALs) and achieves a soluble zymogen expression level of 64 mg L−1 in E. coli, substantially exceeding reported expression levels for natural PALs, while maintaining catalytic efficiency comparable to the fastest known natural PAL, butelase-1 (kcat/Km = 1.3 × 106 M−1 s−1). Notably, the enhanced structural stability does not translate into a corresponding shift in the optimal reaction temperature, highlighting a decoupling between global folding stability and catalytic performance. Together, these results demonstrate how a constrained, diffusion-enabled engineering workflow can transform fragile enzyme scaffolds into robust, application-ready biocatalysts and highlight the importance of integrating generative protein design with experimental optimization to translate in silico designs into high-performance dynamic enzymes.

Related

  1. Selective and Efficient Functionalization of P22 Virus-Like Particles Using an Asparaginyl Ligase.
    Maxim D Harding, Mark A Jackson, Kuok Yap +4
    Bioconjugate chemistryPeer-reviewedCC BY-NC-NDFull text1 citedMolecular biology & gene regulationEurope PMC
  2. Identifying Differentially Expressed Proteins via Plasma Exosomal Proteomics
    Li-Li Zhang, Guo-Wei He
    Bio-protocolPeer-reviewedCC BY-NCFull textMolecular biology & gene regulationEurope PMC
  3. Loss‐of‐function of gynoecium‐expressed phospholipase pPLAIIγ triggers maternal haploid induction in Arabidopsis
    Jin Hoon Jang, Hae Seong Seo, Thomas Widiez +1
    New PhytologistPeer-reviewedHAL deposit licenceFull text31 citedMolecular biology & gene regulationHAL
  4. Fzd7 (Frizzled-7) Expressed by Endothelial Cells Controls Blood Vessel Formation Through Wnt/β-Catenin Canonical Signaling
    Claire Peghaire, Marie Lise Bats, Raj Sewduth +5
    Arteriosclerosis, Thrombosis, and Vascular BiologyPeer-reviewedLicence not stated36 citedMolecular biology & gene regulationHAL
  5. The NuRD complex shapes RNA polymerase III activity at highly expressed tRNA gene clusters, tuning the dynamic range of cellular tRNA pools
    Ruiying Cheng, Rajendra K C, Simon Lizarazo +4
    Cell ReportsPeer-reviewedCC BY 4.0Molecular biology & gene regulationCrossref

Cite

BibTeXRIS
BibTeX
@article{pang2026constrained,
  title = {{Constrained diffusion-enabled construction of a highly expressed and robust peptide asparaginyl ligase}},
  author = {Shuo Pang and Yuxing Hao and Jiarong Mo and Yingying He and Yinglu Cui and Bian Wu and Xinya Hemu},
  journal = {Chemical Science},
  year = {2026},
  month = sep,
  doi = {10.1039/d6sc04842k},
  url = {https://doi.org/10.1039/d6sc04842k},
  note = {CC BY-NC 3.0},
}
RIS
TY  - JOUR
TI  - Constrained diffusion-enabled construction of a highly expressed and robust peptide asparaginyl ligase
AU  - Shuo Pang
AU  - Yuxing Hao
AU  - Jiarong Mo
AU  - Yingying He
AU  - Yinglu Cui
AU  - Bian Wu
AU  - Xinya Hemu
PY  - 2026
DA  - 2026/09/15
JO  - Chemical Science
DO  - 10.1039/d6sc04842k
UR  - https://doi.org/10.1039/d6sc04842k
AB  - Peptide asparaginyl ligases (PALs) hold great promise for peptide macrocyclization and protein bioconjugation, yet their broader application is limited by insufficient thermal stability and low recombinant expression. Here, we report FortiPAL-1, a diffusion-enabled PAL developed through an integrated computational-experimental strategy that couples de novo sequence generation with multi-model-guided mutagenesis and PAL-specific functional refinements. Compared with natural PALs, FortiPAL-1 exhibits markedly enhanced thermal stability (Tm = 69 °C versus ∼50 °C for typical PALs) and achieves a soluble zymogen expression level of 64 mg L−1 in E. coli, substantially exceeding reported expression levels for natural PALs, while maintaining catalytic efficiency comparable to the fastest known natural PAL, butelase-1 (kcat/Km = 1.3 × 106 M−1 s−1). Notably, the enhanced structural stability does not translate into a corresponding shift in the optimal reaction temperature, highlighting a decoupling between global folding stability and catalytic performance. Together, these results demonstrate how a constrained, diffusion-enabled engineering workflow can transform fragile enzyme scaffolds into robust, application-ready biocatalysts and highlight the importance of integrating generative protein design with experimental optimization to translate in silico designs into high-performance dynamic enzymes.
SN  - 2041-6520
SN  - 2041-6539
ER  -