中科院上海有机化学研究所刘文团队揭示，Linaridin途径的重建提供了在结构低估的Cypemycin形成的两种膜相关蛋白的家族决定活性的途径。相关研究成果于2023年3月15日发表在国际顶尖学术期刊《美国化学会杂志》。

Cypemycin是一种已知含有非蛋白原性脱氢丁酸、N，N-二甲基丙氨酸和氨基乙烯基半胱氨酸残基的linaridin亲本肽。形成这种核糖体合成肽的酶促过程仍然难以捉摸，这主要是因为缺乏对由CypH和CypL进行的翻译后修饰（PTM）的了解，这两种膜相关酶是linaridin生物合成所特有的。

基于天蓝色链霉菌中该途径的异源重组，研究人员报道了作为一种以前未知的富含d-氨基酸的环丙啶的cypemycin的详细结构特征。特别是，揭示了CypH和CypL前所未有的家族决定活性，除了水解以去除N-末端前导肽外，还通过残基差向异构化（11个氨基酸）、脱水（4-Thr）和脱硫（Cys19）的机制相关的16个反应，导致前体肽的核心肽部分转化。

随后的linaridin成熟的功能化包括CypD参与的氨基乙烯基半胱氨酸的形成和需要CypM活性的新暴露的N-末端d-Ala残基的N，N-二甲基化。使用遗传、化学、生物化学、工程和建模方法来获得cypemycin的结构以及在催化中实现的CypH和CypL组合的多功能性。

该项工作进一步提高了对PTM化学的认识，并促进了利用合成生物学方法扩大linaridin结构多样性的研究。

附：英文原文

Title: Reconstitution of the Linaridin Pathway Provides Access to the Family-Determining Activity of Two Membrane-Associated Proteins in the Formation of Structurally Underestimated Cypemycin

Author: Yanqing Xue, Xiaofeng Wang, Wen Liu

Issue&Volume: March 15, 2023

Abstract: Cypemycin is a parent linaridin peptide known to contain nonproteinogenic dehydrobutyrine, N,N-dimethylalanine, and aminovinyl-cysteine residues. The enzymatic process by which this ribosomally synthesized peptide is formed remains elusive largely because of the deficiency of knowledge in post-translational modifications (PTMs) conducted by CypH and CypL, the two membrane-associated enzymes unique to linaridin biosynthesis. Based on heterologous reconstitution of the pathway in Streptomyces coelicolor, we report the detailed structural characterization of cypemycin as a previously unknown, d-amino acid-rich linaridin. In particular, the unprecedented family-determining activity of CypH and CypL was revealed, which, in addition to hydrolysis for removal of the N-terminal leader peptide, leads to transformation of the core peptide part of the precursor peptide through mechanistically related 16 reactions for residue epimerization (11 amino acids), dehydration (4 Thr), and dethiolation (Cys19). Subsequent functionalization for linaridin maturation includes CypD-involved aminovinyl-cysteine formation and N,N-dimethylation of the newly exposed N-terminal d-Ala residue that requires CypM activity. Genetic, chemical, biochemical, engineering, and modeling approaches were used to access the structure of cypemycin and the versatility of the CypH and CypL combination that is achieved in catalysis. This work furthers the appreciation of PTM chemistry and facilitates efforts for expanding linaridin structural diversity using synthetic biology methods.

DOI: 10.1021/jacs.3c01730

Source: https://pubs.acs.org/doi/10.1021/jacs.3c01730