A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

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 k cat /K m 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.

Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

“…3). As demonstrated here and in previous works, 27,37 AEP functions from pH $5.0-7.5, but some proteins may become unstable even under mildly acidic environments. Hence, the presented labeling system was also tested with the engineered lumazine synthase AaLS-13, a macromolecular complex composed of 360 protein subunits which is prone to precipitation at pH below 7.0.…”

“…Protein-based approaches offer the ability to function efficiently under mild reaction conditions. 5,6 Transferase, [13][14][15] oxidoreductase, [16][17][18] ligase, 19,20 transpeptidase 2,4,[21][22][23][24][25][26][27] and (split-)intein [28][29][30] have been applied for protein bioconjugation. Nevertheless, adapting these enzymes or proteins for synthetic applications, which deviate from their natural function, oen results in technical issues.…”

Structural basis for a natural circular permutation in proteins

“…The structural features that make AEP favour hydrolysis or transpeptidation remains a matter of debate. Recent work by Du et al (2020) uncovered a plant AEP with one of the highest transpeptidase activities despite predictions, based on previously published AEPs, that it would be a hydrolase. Here we compared CeAEP1 structure to butelase1, an extremely efficient transpeptidase with efficiency up to 1,340,000 M -1 s -1 , and to VyPAL2, OaAEP1, AtLegγ, and HaAEP1, which have intermediate to low transpeptidase efficiency.…”

An environmentally sustainable biomimetic production of cyclic disulfide-rich peptides

“…31 A disadvantage of sortase and trypsiligase is their strict recognition sequence requirements, whereas subtiligase requires a C-terminal carboxamidomethyl ester on the CP. AEPs are highly efficient at catalyzing backbone cyclization of linear peptides, 32,33 rendering them attractive tools for chemoenzymatic cyclization. AEPs require a simple tripeptide Asx-Xaa-Yaa (P1-P1ʹ-P2ʹ) recognition motif, where Asx is Asn or Asp in position P1 followed by any small residue in P1ʹ and a hydrophobic or aliphatic amino acid in P2ʹ.…”

“…One approach to overcome this limitation is intein-mediated backbone cyclization but the requirement for a reduced cysteine at the cyclization/processing sites may be problematic for certain peptides. 37,39,40 AEPs are ideally suited for cyclization applications because they are highly efficient at catalyzing backbone cyclization of naturally occurring cyclic peptides 32,33 and require minimal recognition sequences. 34 Poon et al demonstrated that cyclic peptides can be produced in plant-based expression systems by co-expressing an asparaginyl endopeptidase (AEP) with a peptide precursor equipped with a suitable cyclization motif.…”