Scholarlink

<p>Peptide asparaginyl ligases (PALs) are powerful tools for protein engineering but are limited by natural rarity and poor expression. We mine 23 cyclotide-rich <i>Viola</i> 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 <i>wt-</i>VyPAL2 and 12 mg L<sup>-1</sup> soluble expression in <i>E. coli</i>. 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.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年4月27日

查看全文
  • Wenyu Du,
  • Shi Qi,
  • Mingming Zhen,
  • Miao Liao,
  • Yang Zhao,
  • Jiahui Ma,
  • Yining Hao,
  • Hui Jiang,
  • Gengyu Lu,
  • Chong Wai Liew,
  • Niying Chua,
  • Huachao Chen,
  • Guangwan Hu,
  • Xinya Hemu

摘要

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.