Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States.
ACS Chem Biol. 2023 Aug 18;18(8):1713-1718. doi: 10.1021/acschembio.3c00328. Epub 2023 Aug 9.
Nitration reactions are crucial for many industrial syntheses; however, current protocols lack site specificity and employ hazardous chemicals. The noncanonical cytochrome P450 enzymes RufO and TxtE catalyze the only known direct aromatic nitration reactions in nature, making them attractive model systems for the development of analogous biocatalytic and/or biomimetic reactions that proceed under mild conditions. While the associated mechanism has been well-characterized in TxtE, much less is known about RufO. Herein we present the first structure of RufO alongside a series of computational and biochemical studies investigating its unusual reactivity. We demonstrate that free l-tyrosine is not readily accepted as a substrate despite previous reports to the contrary. Instead, we propose that RufO natively modifies l-tyrosine tethered to the peptidyl carrier protein of a nonribosomal peptide synthetase encoded by the same biosynthetic gene cluster and present both docking and molecular dynamics simulations consistent with this hypothesis. Our results expand the scope of direct enzymatic nitration reactions and provide the first evidence for such a modification of a peptide synthetase-bound substrate. Both of these insights may aid in the downstream development of biocatalytic approaches to synthesize rufomycin analogues and related drug candidates.
硝化反应在许多工业合成中至关重要；然而，目前的方法缺乏位点特异性，并且使用危险化学品。非典型细胞色素 P450 酶 RufO 和 TxtE 催化自然界中唯一已知的直接芳香硝化反应，使它们成为开发类似生物催化和/或仿生反应的有吸引力的模型系统，这些反应在温和条件下进行。虽然 TxtE 中的相关机制已经得到很好的描述，但关于 RufO 的了解要少得多。本文介绍了 RufO 的第一个结构以及一系列计算和生化研究，以研究其不寻常的反应性。我们证明，尽管之前有报道称并非如此，但游离 l-酪氨酸不易被接受为底物。相反，我们提出 RufO 天然修饰与非核糖体肽合成酶的肽酰载体蛋白连接的 l-酪氨酸，该非核糖体肽合成酶由相同的生物合成基因簇编码，并呈现与该假设一致的对接和分子动力学模拟。我们的结果扩展了直接酶促硝化反应的范围，并提供了第一个证据证明肽合成酶结合的底物发生了这种修饰。这两个见解都可能有助于开发生物催化方法来合成 rufomycin 类似物和相关药物候选物。
