Jennifer Teo

In the asparaginyl endopeptidase (AEP) family, only a few members were discovered to acquire ligase activity. Through data mining, AEP candidates with similar sequences and identical catalytic triad as butelase 1 can be found in Viola uliginosa (Nguyen, 2014). VuAL, the plant ligase isolated from Viola uliginosa, can catalyze the backbone ligation of peptide and protein precursors. This enables peptide macrocyclization and synthesis. VuAL-mediated ligation can potentially play an important role in future pharmaceutical and bioengineering applications. These are namely cyclization of therapeutic proteins, labelling of live cells and viral capsids, protein immobilization to solid support, macromolecular conjugation, tandem protein ligation and protein-protein fusion. However, the ligation efficiency of VuAL is still much weaker as compared to butelase 1. Research has proven that the mutation of a single residue in the sequence can significantly boost its catalytic efficiency (Yang, 2017). It is believed that through the alteration of an Isoleucine to an Alanine could render a less bulky conformation near the catalytic triad, providing more space and thus easier for the substrate to contact with the active site. Therefore, we aim to investigate and compare VuAL I247A with VuAL in terms of their ligase efficiency.