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: Nerium oleander and Thevetia peruviana of the family Apocynaceae; Bauhinia variegata, Dermatophyllum secundiflorum, Pithecellobium flexicaule, and Prosopis chilensis of the family Fabaceae; Morus alba of the family Moraceae; and Citrus aurantium 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 D. secundiflorum leaves showed that the yield of cyclic peptide remained stable around 0.5 ± 0.1% of the substrate over the course of the reaction.
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We thank Rizal F. Hariadi (Arizona State University), Edward K. Gilding (The University of Queensland) and Mark A. Jackson (The University of Queensland) for the helpful discussion, Gabrielle R. Hirneise (Arizona State University) for help with the manuscript, and the Biodesign Institute, Arizona State University, for access to their mass spectrometry facility.
This study was supported by the Arizona State University Start-Up Funds to Rizal F. Hariadi and Nicholas Stephanopoulos.
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
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Mahatmanto, T., Azizah, I., Buchberger, A. et al. Progress toward sourcing plants for new bioconjugation tools: a screening evaluation of a model peptide ligase using a synthetic precursor. 3 Biotech 9, 442 (2019). https://doi.org/10.1007/s13205-019-1983-2
- Plant peptide ligase
- Asparaginyl endopeptidase
- Cyclic peptide
- Protein modification
- Bioconjugation tool