Improved Asparaginyl‐Ligase‐Catalyzed Transpeptidation via Selective Nucleophile Quenching

Abstract

The use of enzymes for the site‐specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP‐catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving‐group peptide. Our metal‐complexation‐based strategy enables efficient peptide/protein labeling at the N‐ or C‐terminus with near‐equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein–protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable.

Enzymatic C‐to‐C Protein Ligation

“…As the initial P1′‐P2′ leaving group is released from the recognition motif over the course of the reaction, this byproduct competes with the desired incoming nucleophile N‐termini, culminating in a product‐limiting equilibrium. By simply extending the Asn‐Gly‐Leu motif by a single His, the nucleophilicity of released Gly‐Leu‐His byproducts can be quenched via metal complex formation upon addition of Ni 2+ to the reaction (Figure 1A) thereby suppressing the reverse reaction [8a] . We hypothesized that this quenching approach could enable the efficient incorporation of suboptimal incoming N‐termini (P1′′‐P2′′) which would otherwise be readily outcompeted by the more efficiently incorporated Gly‐Leu (P1′‐P2′) byproduct.…”

Asparaginyl endopeptidases: enzymology, applications and limitations

“…Quenching of the nucleophilic by-product prevents the reverse peptide ligation by OaAEP1-C247A, thus driving the reaction equilibrium towards product formation. 42 Both of these reports are excellent proof of concepts facilitating protein labelling at the terminus of choice (N or C) with a lowered amount of label. Nevertheless, improvements are still required.…”

“…Excellent kinetic parameters and relatively short recognition sequences indicate that AEP methodologies would be a valuable addition to supplement the existing approaches. AEPs have been employed to modify protein substrates including GFP, 46,47,100 ubiquitin, 37,46,101 ompA, 102 DARPin, 101 maltose binding proteins, 42,47 and nanobodies 40 with a range of synthetic labels including click handles, polyethylene glycol, fluorophores and drug molecules.…”

Plant asparaginyl endopeptidases and their structural determinants of function

“…The broad specificity for any nucleophilic P1″ amino acid except proline [ 6 , 99 ] in the transpeptidation reaction makes AEPs ideal enzymes for a variety of protein or peptide substrates. However, this promiscuity comes at a cost as ingenious workarounds are required to minimise nucleophilic attack by the cleaved by-product [ 92 , 100 , 101 ]. The study and genetic engineering of AEP provides an alternative to the chemical ligation of peptides and is therefore crucial for developing novel macrocyclic peptides for use in agriculture and medicine.…”