Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases
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.
Authors
- Gengyu Lu (ORCID: https://orcid.org/0000-0003-2633-8889)
- Wenyu Du
- Xinya Hemu (ORCID: https://orcid.org/0000-0003-1979-5854)
- Huachao Chen (ORCID: https://orcid.org/0000-0002-0326-3012)
- Mingming Zhen (ORCID: https://orcid.org/0000-0002-0641-957X)
- Niying Chua (ORCID: https://orcid.org/0009-0009-4780-3592)
- Miao Liao (ORCID: https://orcid.org/0000-0003-3378-3817)
- Yining Hao (ORCID: https://orcid.org/0000-0002-4066-048X)
- Chong Wai Liew (ORCID: https://orcid.org/0000-0002-4343-818X)
- Jiahui Ma
- Jiang Hui (ORCID: https://orcid.org/0000-0002-8670-8231)
- Shi Qi
- Yang Zhao
- Guangwan Hu (ORCID: https://orcid.org/0000-0001-7728-7976)
Institutions
- China Pharmaceutical University (CN)
- Nanyang Technological University (SG)
- Chinese Academy of Sciences (CN)
- Jianghan University (CN)
- Wuhan Botanical Garden (CN)
- Experimental Drug Development Centre (SG)
- Zhejiang Pharmaceutical College (CN)
- University of Chinese Academy of Sciences (CN)
Publication Details
- Journal
- Nature Communications
- Published
- 2026-04-27
- DOI
- https://doi.org/10.1038/s41467-026-72367-y
- Primary Topic
- Biochemical and Structural Characterization
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- National Natural Science Foundation of China
- National Key Research and Development Program of China