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Updated: Jan 24, 2026
14:44
Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Published on: December 16, 2013
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Structural determinants for peptide-bond formation by asparaginyl ligases
Xinya Hemu1, Abbas El Sahili1,2, Side Hu1,2
1School of Biological Sciences, Nanyang Technological University, 637551 Singapore.
Summary
Plant enzymes called asparaginyl endopeptidases (AEPs) can function as peptide asparaginyl ligases (PALs) by forming bonds instead of breaking them. Structural determinants, LAD1 and LAD2, control this ligase activity, enabling enzyme engineering.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Asparaginyl endopeptidases (AEPs) are cysteine proteases that cleave Asx-Xaa bonds under acidic conditions.
- Certain plant enzymes, like butelase 1, function as peptide asparaginyl ligases (PALs), catalyzing bond formation in near-neutral conditions and acting as macrocyclases for cyclic peptide biosynthesis.
Purpose of the Study:
- To elucidate the mechanism by which PALs function as ligases rather than proteases.
- To identify the structural determinants responsible for ligase activity in AEPs from the Violaceae family.
Main Methods:
- Sequence homology analysis to identify AEPs and PALs from *Viola yedoensis* and *Viola canadensis*.
- X-ray crystallography of a PAL at 2.4-Å resolution.
- Site-directed mutagenesis studies on identified enzymes.
Main Results:
- Ligase-activity determinants, LAD1 and LAD2, flanking the S1 site were discovered, modulating activity by controlling nucleophile access to the S-ester intermediate.
- Recombinant expression and functional studies confirmed *Vy*PAL1-3 as ligases.
- Mutagenesis of LAD1 and LAD2 in *Vy*PAL1-3, *Vy*AEP1, and *Vc*AEP supported their role in ligase activity, with LAD2 mutagenesis converting a protease (*Vc*AEP) into a ligase.
Conclusions:
- LAD1 and LAD2 are crucial structural determinants for peptide asparaginyl ligase activity.
- Understanding these determinants facilitates the genomic identification of PALs and the engineering of AEPs into PALs.
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