Edited by Stephen J. Benkovic, The Pennsylvania State University, University Park, PA, and approved March 12, 2019 (received for review February 5, 2019)
Ribosomally synthesized and post-translationally modified peptides are natural products that hold great promise for a range of medical and biotechnological applications. However, the cost-effective heterologous production of these peptides is hampered by a poor understanding of their biosynthesis. Cyclotides, plant-derived, disulfide-knotted, head-to-tail cyclic peptides, exhibit exceptional stability and great amenability for amino acid substitutions and insertions. Although much effort has been invested toward understanding cyclotide biosynthesis, details of the key proteolytic step before cyclization remained elusive for two decades. We used an activity-guided approach to discover the enzymes involved. Our characterization of these enzymes will enable efficient approaches for heterologous cyclotide production.
Cyclotides are plant defense peptides that have been extensively investigated for pharmaceutical and agricultural applications, but key details of their posttranslational biosynthesis have remained elusive. Asparaginyl endopeptidases are crucial in the final stage of the head-to-tail cyclization reaction, but the enzyme(s) involved in the prerequisite steps of N-terminal proteolytic release were unknown until now. Here we use activity-guided fractionation to identify specific members of papain-like cysteine proteases involved in the N-terminal cleavage of cyclotide precursors. Through both characterization of recombinantly produced enzymes and in planta peptide cyclization assays, we define the molecular basis of the substrate requirements of these enzymes, including the prototypic member, here termed kalatase A. The findings reported here will pave the way for improving the efficiency of plant biofactory approaches for heterologous production of cyclotide analogs of therapeutic or agricultural value.
Author contributions: T.D. and D.J.C. designed research; F.B.H.R., M.A.J., E.D.G., K.Y., E.K.G., and T.D. performed research; F.B.H.R., M.A.J., E.D.G., K.Y., E.K.G., T.D., and D.J.C. analyzed data; and F.B.H.R., M.A.J., E.K.G., T.D., and D.J.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1901807116/-/DCSupplemental.
Published under the PNAS license.
