A suite of kinetically superior AEP ligases can cyclise an intrinsically disordered protein

Abstract

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These “ligase-type” AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1 b . These AEPs work preferentially as ligases at both acidic and neutral pH and we term them “canonical AEP ligases” to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).

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

“…This is consistent with other reports including studies of human and plant legumains that require self-processing at acidic pH to mature into a fully active form 14,24,39 . We also found the autoprocessing sites (Asp47 and Asn335) and arrangement of the putative catalytic triad (Asn71, His176, Cys218) of MCoAEP2 are similar to other previously characterized AEPs from O. affinis 13,40 , C. ternatea 12 , jack bean 41 , sunflower 14 , Viola species 36 , and human 23,24 . Furthermore, we showed recombinantly produced MCoAEP2 not only efficiently cyclized a linear MCoTI-II-DAL precursor but also a grafted cyclic peptide based on the MCoTI-II scaffold.…”

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.…”

“…Many asparaginyl endopeptidases (AEPs) from plants possess transpeptidase function and present as ideal biocatalysts for protein labeling (i.e., intermolecular ligation). 24,26,27,[36][37][38] AEP hydrolyzes the C-terminal amide bond of an internal asparagine or aspartate residue (P1) and subsequently mediate ligation to the N-terminus of an incoming nucleophile peptide (Fig. 1).…”

Target Enzymes Considered for the Treatment of Alzheimer’s Disease and Parkinson’s Disease

“…Ac-ESEN-CHO (acetyl-Glu-Ser-Glu-Asn-aldehyde) is also a specific AEP inhibitor. These two short peptides suppressed AEP activity in the plant leaves [ 123 , 124 ]. Cystatins consist of two groups of cysteine protease inhibitors (type 1 and type 2) that have conserved a sequence as G9, Q53, V55, and G57.…”