Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD–DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC₅₀ = 29 ± 4 μM) through the inhibition of NpsA (K_D = 29 ± 4 nM).

中文翻译：

---

机会性病原体产酸克雷伯菌产生的肠毒素替利霉素的生物合成、作用机制和抑制。

替利霉素是一种由机会性病原体产酸克雷伯菌产生的肠毒素，可引起抗生素相关性出血性结肠炎 (AAHC)。这种吡咯并苯二氮卓 (PBD) 天然产物是通过双模块非核糖体肽合成酶 (NRPS) 途径合成的，该途径由三种蛋白质组成：NpsA、ThdA 和 NpsB。我们描述了完全重构的 NRPS 系统的功能和结构表征，并报告了所有天然底物和辅因子的稳态动力学分析以及 NpsA 和 ThdA 的结构表征。提利霉素的作用机制通过 DNA 加合物组学技术通过检测提利霉素暴露于真核细胞后推定的 N-2 鸟嘌呤烷基化得到证实，这提供了细胞中形成的 PBD-DNA 加合物的第一个结构表征。最后，我们报告了小分子抑制剂的合理设计，通过抑制 NpsA ( K _{D = 29 ± 4 nM) 来阻断全细胞产酸克雷伯菌 (} K. oxytoca ) 中的提利霉素生物合成(IC ₅₀ = 29 ± 4 μM)。