Department of Pharmaceutical Sciences, University of Kentucky , Lexington, Kentucky 40536-0596, United States.
J Am Chem Soc. 2014 Dec 10;136(49):17350-4. doi: 10.1021/ja510489j. Epub 2014 Nov 26.
The antitumor agent thiocoraline is a nonribosomally biosynthesized bisintercalator natural product, which contains in its peptidic backbone two S-methylated l-cysteine residues. S-Methylation occurs very rarely in nature, and is observed extremely rarely in nonribosomal peptide scaffolds. We have proposed that during thiocoraline biosynthesis, TioN, a stand-alone adenylation domain interrupted by the S-adenosyl-l-methionine binding region of a methyltransferase enzyme, is capable of performing two functions: the adenylation and S-methylation of l-cysteine. Herein, by preparation of knockouts of TioN and its MbtH-like protein partner TioT, we confirmed their role in thiocoraline biosynthesis. We also co-expressed recombinant TioN and TioT and biochemically investigated three potential pathways involving activation, methylation, and loading of l-cysteine onto the TioN partner thiolation domain, TioS(T4). The valuable insights gained into the pathway(s) followed for the production of S-Me-l-Cys-S-TioS(T4) will serve as a guide for the development of novel engineered interrupted adenylation enzymes for combinatorial biosynthesis.
抗肿瘤剂硫代珊瑚素是一种非核糖体生物合成的双嵌入剂天然产物，其肽骨干中含有两个 S-甲基化 l-半胱氨酸残基。S-甲基化在自然界中很少发生，在非核糖体肽支架中也很少观察到。我们曾提出，在硫代珊瑚素生物合成过程中，TioN（一个独立的氨酰化结构域，被甲基转移酶的 S-腺苷甲硫氨酸结合区域所打断）能够执行两个功能：l-半胱氨酸的氨酰化和 S-甲基化。在此，通过制备 TioN 及其 MbtH 样蛋白伴侣 TioT 的敲除体，我们证实了它们在硫代珊瑚素生物合成中的作用。我们还共表达了重组 TioN 和 TioT，并对涉及 l-半胱氨酸的激活、甲基化和加载到 TioN 伴侣硫醇化结构域 TioS(T4)的三个潜在途径进行了生物化学研究。对 S-Me-l-Cys-S-TioS(T4)生成所遵循的途径的深入了解，将为组合生物合成中新型工程化的中断氨酰化酶的开发提供指导。
