The Gram-negative selective antibiotic darobactin A has attracted interest owing to its intriguing fused bicyclic structure and unique targeting of the outer membrane protein BamA. Darobactin, a ribosomally synthesized and post-translationally modified peptide (RiPP), is produced by a radical S-adenosyl methionine (rSAM)-dependent enzyme (DarE) and contains one ether and one C–C cross-link. Herein, we analyze the substrate tolerance of DarE and describe an underlying catalytic principle of the enzyme. These efforts produced 51 enzymatically modified darobactin variants, revealing that DarE can install the ether and C–C cross-links independently and in different locations on the substrate. Notable variants with fused bicyclic structures were characterized, including darobactin W3Y, with a non-Trp residue at the twice-modified central position, and darobactin K5F, which displays a fused diether ring pattern. While lacking antibiotic activity, quantum mechanical modeling of darobactins W3Y and K5F aided in the elucidation of the requisite features for high-affinity BamA engagement. We also provide experimental evidence for β-oxo modification, which adds support for a proposed DarE mechanism. Based on these results, ether and C–C cross-link formation was investigated computationally, and it was determined that more stable and longer-lived aromatic Cβ radicals correlated with ether formation. Further, molecular docking and transition state structures based on high-level quantum mechanical calculations support the different indole connectivity observed for ether (Trp-C7) and C–C (Trp-C6) cross-links. Finally, mutational analysis and protein structural predictions identified substrate residues that govern engagement to DarE. Our work informs on darobactin scaffold engineering and further unveils the underlying principles of rSAM catalysis.

中文翻译：

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Darobactin 底物工程和计算显示自由基稳定性控制着醚与 C-C 键的形成


革兰氏阴性选择性抗生素 darobactin A 因其有趣的融合双环结构和对外膜蛋白 BamA 的独特靶向性而引起了人们的兴趣。 Darobactin 是一种核糖体合成的翻译后修饰肽 (RiPP)，由自由基 S-腺苷甲硫氨酸 (rSAM) 依赖性酶 (DarE) 产生，含有一个醚和一个 C-C 交联。在此，我们分析了 DarE 的底物耐受性并描述了该酶的基本催化原理。这些努力产生了 51 种酶促修饰的 Darobactin 变体，表明 DarE 可以独立地在基质的不同位置安装醚和 C-C 交联。对具有稠合双环结构的显着变体进行了表征，包括在两次修饰的中心位置具有非色氨酸残基的darobactin W3Y，以及显示稠合二醚环模式的darobactin K5F。虽然缺乏抗生素活性，darobactins W3Y 和 K5F 的量子力学建模有助于阐明高亲和力 BamA 结合的必要特征。我们还提供了 β-oxo 修饰的实验证据，这增加了对所提出的 DarE 机制的支持。基于这些结果，通过计算研究了醚和 C-C 交联的形成，并确定更稳定和寿命更长的芳香族 Cβ 自由基与醚的形成相关。此外，基于高级量子力学计算的分子对接和过渡态结构支持在醚 (Trp-C7) 和 C-C (Trp-C6) 交联中观察到的不同吲哚连接性。最后，突变分析和蛋白质结构预测确定了控制与 DarE 结合的底物残基。 我们的工作为 Darobactin 支架工程提供了信息，并进一步揭示了 rSAM 催化的基本原理。