%0 Journal Article %A Side Hu %A Abbas El Sahili %A Srujana Kishore %A Yee Hwa Wong %A Xinya Hemu %A Boon Chong Goh %A Zhen Wang %A James P. Tam %A Chuan-Fa Liu %A Julien Lescar %T Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis %D 2021 %R 10.1101/2021.12.09.471967 %J bioRxiv %P 2021.12.09.471967 %X Peptide asparaginyl ligases (PALs) belong to a limited class of enzymes from cyclotide-producing plants, that perform site-specific ligation reactions after a target peptide Asx (Asn/Asp) binds to the ligase active site. How PALs specifically recognize their polypeptide substrates has remained elusive especially at the prime binding side of the enzyme. Here we captured VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme’s S1 pocket and why a hydrophobic residue is required at the substrate P2’ position. Beside illustrating the role played by P1 and P2’ residues as primary anchors for the enzyme reaction, these results provide a mechanistic explanation for the role of the “Gatekeeper” residue at the surface of the S2 pocket, in shifting the non-prime portion of the substrate and, as a result, the activity towards either ligation or hydrolysis. These results detail the molecular events that occur during proenzyme maturation in the plant vacuolar compartment, suggest a mechanism for ligation, and will inform the design of peptide ligases with tailored specificities.One sentence summary We captured VyPAL2, a catalytically efficient plant peptide ligase with a bound substrate, providing the molecular basis for substrate recognition and ligation. %U https://www.biorxiv.org/content/biorxiv/early/2021/12/09/2021.12.09.471967.full.pdf