State Key Laboratory of Coordination Chemistry, Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Nanjing University, 210023 Nanjing, China.
State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, 210023 Nanjing, China.
Proc Natl Acad Sci U S A. 2019 Feb 12;116(7):2533-2538. doi: 10.1073/pnas.1815594116. Epub 2019 Jan 24.
Lanthipeptides are an important subfamily of ribosomally synthesized and posttranslationally modified peptides, and the removal of their N-terminal leader peptides by a designated protease(s) is a key step during maturation. Whereas proteases for class I and II lanthipeptides are well-characterized, the identity of the protease(s) responsible for class III leader processing remains unclear. Herein, we report that the class III lanthipeptide NAI-112 employs a bifunctional Zn-dependent protease, AplP, with both endo- and aminopeptidase activities to complete leader peptide removal, which is unprecedented in the biosynthesis of lanthipeptides. AplP displays a broad substrate scope in vitro by processing a number of class III leader peptides. Furthermore, our studies reveal that AplP-like proteases exist in the genomes of all class III lanthipeptide-producing strains but are usually located outside the biosynthetic gene clusters. Biochemical studies show that AplP-like proteases are universally responsible for the leader removal of the corresponding lanthipeptides. In addition, AplP-like proteases are phylogenetically correlated with aminopeptidase N from , and might employ a single active site to catalyze both endo- and aminopeptidyl hydrolysis. These findings solve the long-standing question as to the mechanism of leader peptide processing during class III lanthipeptide biosynthesis, and pave the way for the production and bioengineering of this class of natural products.
硫肽是一类重要的核糖体合成和翻译后修饰肽，其 N 端前导肽被特定蛋白酶（s）切除是成熟过程中的关键步骤。尽管 I 型和 II 型硫肽的蛋白酶已得到很好的研究，但负责 III 型前导肽加工的蛋白酶的身份仍不清楚。在此，我们报告 III 型硫肽 NAI-112 采用具有内切酶和氨肽酶活性的双功能 Zn 依赖性蛋白酶 AplP 完成前导肽切除，这在硫肽生物合成中是前所未有的。AplP 在体外通过处理多种 III 型前导肽显示出广泛的底物范围。此外，我们的研究表明，AplP 样蛋白酶存在于所有 III 型硫肽产生菌株的基因组中，但通常位于生物合成基因簇之外。生化研究表明，AplP 样蛋白酶普遍负责相应硫肽的前导肽去除。此外，AplP 样蛋白酶与来自 的氨肽酶 N 在系统发育上相关，可能采用单一活性位点催化内肽和氨肽水解。这些发现解决了 III 型硫肽生物合成过程中前导肽加工机制的长期问题，并为该类天然产物的生产和生物工程铺平了道路。
