One enzyme with two functions : how to train proteases to perform ligation

Abstract

Cyclotides, a class of plant-derived cysteine-rich cyclic proteins, are an attractive framework for drug design. Asparaginyl endopeptidases (AEPs) are enzymes that are associated with proteolysis, but there is a class of AEPs that catalyze the backbone cyclization of cyclotide precursors. Here, we report on how to train the bifunctional AEP McPAL-1 to carry out ligation by changing influencing factors. We found that AEPs are substrate specific and the P2” residue affects the directionality of McPAL-1’s activity. The P1 residue and pH is also a determinant of AEP activity. Additionally, we note that the C-terminus of substrates play an important role in catalyzing enzyme reactions. We also made the discovery that previously thought to be ligase-type and protease-type AEPs, are actually all bifunctional in activity.