Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
J Am Chem Soc. 2017 Dec 27;139(51):18623-18631. doi: 10.1021/jacs.7b10203. Epub 2017 Dec 15.
The past decade has seen the discovery of four different classes of radical S-adenosylmethionine (rSAM) methyltransferases that methylate unactivated carbon centers. Whereas the mechanism of class A is well understood, the molecular details of methylation by classes B-D are not. In this study, we present detailed mechanistic investigations of the class C rSAM methyltransferase TbtI involved in the biosynthesis of the potent thiopeptide antibiotic thiomuracin. TbtI C-methylates a Cys-derived thiazole during posttranslational maturation. Product analysis demonstrates that two SAM molecules are required for methylation and that one SAM (SAM1) is converted to 5'-deoxyadenosine and the second SAM (SAM2) is converted to S-adenosyl-l-homocysteine (SAH). Isotope labeling studies show that a hydrogen is transferred from the methyl group of SAM2 to the 5'-deoxyadenosine of SAM1 and the other two hydrogens of the methyl group of SAM2 appear in the methylated product. In addition, a hydrogen appears to be transferred from the β-position of the thiazole to the methyl group in the product. We also show that the methyl protons in the product can exchange with solvent. A mechanism consistent with these observations is presented that differs from other characterized radical SAM methyltransferases.
过去十年见证了四类不同的活性 S-腺苷甲硫氨酸（rSAM）甲基转移酶的发现，它们可以甲基化未激活的碳中心。虽然 A 类的机制已经很清楚，但 B-D 类的甲基化分子细节尚不清楚。在这项研究中，我们对涉及强效硫肽抗生素硫霉素生物合成的 C 类 rSAM 甲基转移酶 TbtI 进行了详细的机制研究。TbtI 在翻译后成熟过程中 C-甲基化 Cys 衍生的噻唑。产物分析表明，甲基化需要两个 SAM 分子，一个 SAM（SAM1）转化为 5'-脱氧腺苷，第二个 SAM（SAM2）转化为 S-腺苷-L-同型半胱氨酸（SAH）。同位素标记研究表明，SAM2 的甲基中的一个氢转移到 SAM1 的 5'-脱氧腺苷上，SAM2 的甲基中的另外两个氢出现在甲基化产物中。此外，似乎有一个氢从噻唑的β位置转移到产物中的甲基上。我们还表明，产物中的甲基质子可以与溶剂交换。提出了一个与其他已描述的自由基 SAM 甲基转移酶不同的符合这些观察结果的机制。
