Abstract
Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a kcat/Km of 620 mM−1 s−1, making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.
Highlights
Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides
We further demonstrate that MCoAEP2 is capable of cyclizing MCoSST-01, an engineered antiangiogenic peptide based on the MCoTI-II scaffold
To characterize AEP-mediated cyclization in vitro, we designed a series of peptide substrates comprising an oxidatively folded cyclotide domain flanked by truncated leader and follower regions (Table 1)
Summary
Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. We report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Cyclotides are a group of plant-derived RiPPs that have a special cyclic cystine knot (CCK) structural motif that confers exceptional stability[3]. Together with their natural function as host defense agents[4], cyclotides have shown promise as scaffolds for biotechnological applications. Asparaginyl endopeptidases (AEPs, known as vacuolar processing enzymes or legumains) have been shown to recognize a conserved Asn/Asp residue at the C-terminal processing site, and cyclize the cyclotide domain by transpeptidation[9,10,11]. Before cyclization, the leader peptide must be removed, and the enzyme(s) involved in this step remain poorly understood
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