Transcriptome-guided discovery and active-site gatekeeper engineering of a Viola arcuata asparaginyl ligase with superior catalytic performance.

Peptide macrocyclization by ligase-type asparaginyl endopeptidases (AEPs) underpins many emerging applications in peptide and protein engineering, yet only a few recombinant ligases currently offer suitable catalytic performance and operational robustness. Here we characterize VaPAL2, a previously unreported AEP from Viola arcuata, and show that its activity can be substantially enhanced through a single gatekeeper substitution. The engineered variant, VaPAL2(I244A), functions as a highly efficient peptide ligase, displaying rapid macrocyclization of GN-type substrates, strong ligation bias on branched sequences, and markedly improved tolerance to organic cosolvents such as 20% DMSO. Notably, VaPAL2(I244A) retains its high activity at near-neutral pH, making it compatible with preparative and protein-compatible conditions. Structural modeling indicates that the I244A substitution modestly widens the S1'-S2 substrate corridor, facilitating nucleophile entry while preserving the catalytic architecture. Together, these findings establish VaPAL2(I244A) as a new, robust macrocyclase that matches or exceeds the performance of widely used recombinant AEP ligase benchmarks under standardized conditions and meaningfully expands the enzymatic toolbox available for chemical biology and peptide engineering.