Xie Xiang-Qian, Guo Wen, Xia Yin-Zheng, Liao Li-Juan, Sun Meng-Xin, Wang Jing-Xue, Gao Jiang-Tao, Yao Hong-Wei, Wang Huan
State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center of Nanjing University, Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, 350002 Fuzhou, China.
ACS Cent Sci. 2025 Jun 18;11(7):1178-1188. doi: 10.1021/acscentsci.5c00569. eCollection 2025 Jul 23.
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 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.
2-氨基乙烯基-半胱氨酸（AviCys）基序代表了一类独特的大环结构，存在于许多核糖体合成及翻译后修饰的肽（RiPPs）中。尽管它们在生物活性中起着至关重要的作用，但其生物合成机制，特别是催化Avi-(Me)-Cys大环化的环化酶，尚未得到充分表征。在此，我们报告了V类羊毛硫肽rosin A1 - A3的发现及生物合成解析，其特征在于一个羊毛硫氨酸（Lan）大环和一个C端2-氨基乙烯基-3-甲基-半胱氨酸（AviMeCys）大环。Rosins促进人包皮成纤维细胞（HSF）迁移，这代表了具有促进细胞迁移活性的羊毛硫肽的首个实例。全面的重组实验表明，区域和立体选择性的AviMeCys大环化由RosX催化，RosX是一种新鉴定的具有激酶样折叠的环化酶。因此，最初被错误注释为激酶样蛋白的RosX样环化酶代表了一类独特的环化酶，其利用烯硫醇基团进行AviCys/AviMeCys大环化。此外，我们证明rosins中Lan的形成遵循底物控制的环化途径，由激酶RosK和裂合酶RosY的复合物实现动力学加速，这与AviMeCys大环化不同。本研究解决了酶促AviCys大环化长期存在的模糊问题，并为跨RiPP亚家族对含AviCys天然产物进行生物合成探索和生物工程提供了基础。
