Progress Toward Sourcing Plants for New Bioconjugation Tools: a Screening Evaluation of a Model Peptide Ligase Using a Synthetic Precursor

In the present study, leaves from 39 phylogenetically distant plant species were sampled and screened for asparaginyl endopeptidase ligase activity using mass spectrometry to test the generality of peptide ligases in plants. A modified version of the sunflower trypsin inhibitor-1 precursor was used as the substrate for reactions with leaf crude extracts and protein fractions. Masses consistent with products of asparaginyl endopeptidase activities that cleave and ligate the substrate into cyclic peptide following the reactions were detected in 8 plants: and of the family Apocynaceae; , , , and of the family Fabaceae; of the family Moraceae; and of the family Rutaceae. This screening result represents a 20% hit rate for finding asparaginyl endopeptidase ligase activity from the arbitrary plants sampled. Analysis following a 2-h reaction of the substrate with the crude extract of leaves showed that the yield of cyclic peptide remained stable around 0.5 ± 0.1% of the substrate over the course of the reaction.