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

## TL;DR

This study identifies a new class of enzymes that help create unique chemical structures in peptides, which could lead to new bioactive compounds.

## Contribution

The discovery of a kinase-fold cyclase, RosX, that catalyzes AviCys macrocyclization in lanthipeptides.

## Key findings

- RosX catalyzes regio- and stereoselective AviMeCys macrocyclization using an enethiol group.
- Rosins promote human foreskin fibroblast cell migration, a novel activity for lanthipeptides.
- Lan formation in rosins follows a substrate-controlled pathway with kinetic assistance from RosK and RosY.

## Abstract

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 in vitro 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.