Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States.
J Am Chem Soc. 2020 Jul 29;142(30):13170-13179. doi: 10.1021/jacs.0c05639. Epub 2020 Jul 16.
Thiopeptides are a broad class of macrocyclic, heavily modified peptide natural products that are unified by the presence of a substituted, nitrogen-containing heterocycle core. Early work indicated that this core might be fashioned from two dehydroalanines by an enzyme-catalyzed aza-[4 + 2] cycloaddition to give a cyclic-hemiaminal intermediate. This common intermediate could then follow a reductive path toward a dehydropiperidine, as in the thiopeptide thiostrepton, or an aromatization path to yield the pyridine groups observed in many other thiopeptides. Although several of the enzymes proposed to perform this cycloaddition have been reconstituted, only pyridine products have been isolated and any hemiaminal intermediates have yet to be observed. Here, we identify the conditions and substrates that decouple the cycloaddition from subsequent steps and allow interception and characterization of this long hypothesized intermediate. Transition state modeling indicates that the key amide-iminol tautomerization is the major hurdle in an otherwise energetically favorable cycloaddition. An anionic model suggests that deprotonation and polarization of this amide bond by TbtD removes this barrier and provides a sufficient driving force for facile (stepwise) cycloaddition. This work provides evidence for a mechanistic link between disparate cyclases in thiopeptide biosynthesis.
噻唑肽是一大类具有广泛结构的、高度修饰的肽类天然产物,它们通过含有取代的含氮杂环核心而统一。早期的工作表明,这个核心可能是由两个脱羟丙氨酸通过酶促的氮杂-[4 + 2]环加成反应形成的,生成一个环状半亚胺中间体。这个常见的中间体可以通过还原途径形成脱水哌啶,就像噻唑肽硫链丝菌素一样,也可以通过芳香化途径生成许多其他噻唑肽中观察到的吡啶基团。尽管已经重新构建了几种被提议执行这种环加成反应的酶,但只分离出了吡啶产物,任何半亚胺中间体都尚未被观察到。在这里,我们确定了使环加成反应与后续步骤解耦的条件和底物,并允许拦截和表征这个长期假设的中间体。过渡态建模表明,酰胺-亚氨基互变异构是在其他能量有利的环加成反应中主要的障碍。阴离子模型表明,TbtD 对这个酰胺键的去质子化和极化消除了这个障碍,并为易于(逐步)环加成提供了足够的驱动力。这项工作为噻唑肽生物合成中不同环化酶之间的机制联系提供了证据。