A Unique Class of Cyclases with a Kinase Fold Catalyzes Enethiol-Mediated Macrocyclization of Aminovinyl-Cysteine Motifs in Lanthipeptides

2-Aminovinyl-cysteine (AviCys) motifs represent a unique class of macrocyclic structures found in many ribosomally synthesized and post-translationally modified peptides (RiPPs). Despite their essential role in bioactivity, their biosynthetic machinery, particularly the cyclases catalyzing Avi-(Me)-Cys macrocyclization, has not been fully characterized. Herein, we report the discovery and biosynthetic elucidation of class V lanthipeptides rosin A1-A3, which feature a lanthionine (Lan) macrocycle and a C-terminal 2-aminovinyl-3-methyl-cysteine (AviMeCys) macrocycle. Rosins promote the migration of human foreskin fibroblast (HSF) cells, representing the first examples of lanthipeptides with cell migration-promoting activity. Comprehensive <i>in vitro</i> reconstitution revealed that the regio- and stereoselective AviMeCys macrocyclization is catalyzed by RosX, a newly identified cyclase with a kinase-like fold. Therefore, RosX-like cyclases, originally misannotated as kinase-like proteins, represent a unique class of cyclases that utilize the enethiol group for AviCys/AviMeCys macrocyclization. Furthermore, we demonstrate that Lan formation in rosins follows a substrate-controlled cyclization pathway with kinetic acceleration by the complex of kinase RosK and lyase RosY, which is distinct from the AviMeCys macrocyclization. This study resolves the long-standing ambiguity of enzymatic AviCys macrocyclization and provides a basis for biosynthetic exploration and bioengineering of AviCys-containing natural products across RiPP subfamilies.