Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina 27710, United States.
Institute for Insect Biotechnology, Justus-Liebig-University of Giessen, Ohlebergsweg 12, 35392 Giessen, Germany.
J Am Chem Soc. 2022 Oct 19;144(41):18876-18886. doi: 10.1021/jacs.2c05565. Epub 2022 Oct 4.
Darobactin A is a ribosomally synthesized, post-translationally modified peptide (RiPP) with potent and broad-spectrum anti-Gram-negative antibiotic activity. The structure of darobactin A is characterized by an ether and C-C crosslinking. However, the specific mechanism of the crosslink formation, especially the ether crosslink, remains elusive. Here, using in vitro enzyme assays, we demonstrate that both crosslinks are formed by the DarE radical -adenosylmethionine (SAM) enzyme in an O-dependent manner. The relevance of the observed activity to darobactin A biosynthesis was demonstrated by proteolytic transformation of the DarE product into darobactin A. Furthermore, DarE assays in the presence of O or [O]water demonstrated that the oxygen of the ether crosslink originates from O and not from water. These results demonstrate that DarE is a radical SAM enzyme that uses oxygen as a co-substrate in its physiologically relevant function. Since radical SAM enzymes are generally considered to function under anaerobic environments, the discovery of a radical SAM oxygenase represents a significant change in the paradigm and suggests that these radical SAM enzymes function in aerobic cells. Also, the study revealed that DarE catalyzes the formation of three distinct modifications on DarA; ether and C-C crosslinks and α,β-desaturation. Based on these observations, possible mechanisms of the DarE-catalyzed reactions are discussed.
达罗巴汀 A 是一种核糖体合成的、翻译后修饰的肽（RiPP），具有强大而广谱的抗革兰氏阴性抗生素活性。达罗巴汀 A 的结构特点是存在醚键和 C-C 交联。然而，交联形成的具体机制，特别是醚键交联，仍然难以捉摸。在这里，我们使用体外酶促实验证明，两种交联都是由 DarE 自由基 - 腺苷甲硫氨酸（SAM）酶以 O 依赖性方式形成的。DarE 产物转化为达罗巴汀 A 的蛋白水解转化证明了观察到的活性与达罗巴汀 A 生物合成的相关性。此外，在存在 O 或 [O]水的情况下进行的 DarE 分析表明，醚键交联的氧源来自 O，而不是水。这些结果表明，DarE 是一种自由基 SAM 酶，在其生理相关功能中使用氧气作为共底物。由于自由基 SAM 酶通常被认为在厌氧环境下发挥作用，因此发现一种自由基 SAM 加氧酶代表了范式的重大改变，并表明这些自由基 SAM 酶在需氧细胞中发挥作用。此外，该研究揭示了 DarE 催化达罗巴汀 A 上三种不同修饰的形成；醚键和 C-C 交联以及 α,β-去饱和作用。基于这些观察结果，讨论了 DarE 催化反应的可能机制。
