Functional Characterization of Cross-Link Formation in Atropopeptide P450 Synthases

Biaryl cross-link-containing peptides make up an emerging class of ribosomally synthesized and post-translationally modified peptides (RiPPs). The C–C, C–N, and C–O cross-links in atropopeptides are installed by atropopeptide P450 synthases. In this work, we characterized three unknown atropopeptide P450 synthases (KutB, KutC, and NteB). Functional characterization of KutC and NteB provided the first in vitro analysis of atropopeptide P450 synthases and revealed that NteB generates both bridge-above and bridge-below atropisomers. Furthermore, docking models of KutB, KutC, and NteB, together with mutational analysis, suggested putative substrate recognition residues. This study provides important insights into the biosynthesis of atropopeptides.