Asparaginyl endopeptidase : a multifaceted enzyme for making and breaking peptide bonds

• Main Author: Liew, Heng Tai
• Other Authors: James P Tam
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Science::Biological sciences::Biochemistry
• Online Access: https://hdl.handle.net/10356/154929
• Institution: Nanyang Technological University

Asparaginyl endopeptidases (AEPs), or legumains, are Asn/Asp (Asx)-specific proteases. Plant AEPs have multifaceted roles in nature, including seed storage protein maturation and cyclic peptides production. They are also useful biological tools for precision modification and biomanufacturing of peptides and proteins. Besides, substrate-binding pockets and pH have been shown recently as the major factors affecting the AEP's ligase and protease activity. However, how the substrates affect the AEP's enzymatic directionality is still poorly understood. Here, we have shown that protease-AEPs could perform ligation using specific peptide substrates. This study is important as it would expand the utility of these AEPs in biotechnological applications and highlight the need to elucidate ligation mechanisms in AEPs. Besides, we have also discovered and characterized a highly stable Asx-ligase displaying a pH-dependent enzymatic profile from Momordica cochinchinensis. McPAL1 catalyzes both P1-Asp ligation and P1-Asn hydrolysis from pH 4 to 6, but predominantly Asn-ligation at pH 6.5-8, making it a valuable tool for peptide and protein engineering and cell surface modification.