Peptide Recognition Sequence Guides Catalytic Side Chain Cross-Linking of Plant Peptides by Copper-Dependent Cyclases

Side chain cross-linking of amino acids is a challenging oxidative enzymatic reaction that has largely been confined to heme and iron sulfur cluster containing enzymes. However, the recent discovery of plant BURP-domain peptide cyclases (BpCs) responsible for the biosynthesis of burpitide natural products has demonstrated that copper-dependent enzymes are able to generate similar cross-links. For example, ArbB2 is responsible for the formation of a Tyr-phenol-O to Leu-C_β bond observed in arabipeptin A, a cyclopeptide alkaloid isolated from the well-known plant Coffea arabica. To investigate this intriguing enzyme family in more detail, we developed minimal peptide substrates for ArbB2, which enabled quantitative studies. By examining reductant dependence, we achieved catalytic turnover for the first time in this enzyme family, which allowed for kinetic, mutational, substrate scope, and multicore processing analyses. Additionally, we established the dioxygen dependence and confirmed the absence of hydrogen peroxide as a side product in the catalytic system. Finally, we extended our study to other BpCs involved in cyclopeptide alkaloid biosynthesis, demonstrating that our findings apply across additional members of this enzyme family. Ultimately, this work provides fundamental insights into a new, widespread family of copper-dependent peptide cyclases and lays the groundwork for future mechanistic investigations.