Papain-like cysteine proteases prepare plant cyclic peptide precursors for cyclization.

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.

5-Hydroxy-pyrrolone based building blocks as maleimide alternatives for protein bioconjugation and single-site multi-functionalization.

Recent dramatic expansion in potential uses of protein conjugates has fueled the development of a wide range of protein modification methods; however, the desirable single-site multi-functionalization of proteins has remained a particularly intransigent challenge. Herein, we present the application of 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2Os) as advantageous alternatives to widely used maleimides for the chemo- and site-selective labeling of cysteine residues within proteins. A variety of 5HP2O building blocks have been synthesized using a one-pot photooxidation reaction starting from simple and readily accessible furans and using visible light and oxygen. These novel reagents display excellent cysteine selectivity and also yield thiol conjugates with superior stability. 5HP2O building blocks offer a unique opportunity to introduce multiple new functionalities into a protein at a single site and in a single step, thus, significantly enhancing the resultant conjugate's properties.