Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States.
Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712, United States.
J Am Chem Soc. 2021 Dec 8;143(48):20291-20295. doi: 10.1021/jacs.1c09482. Epub 2021 Nov 23.
The catalog of enzymes known to catalyze the nucleophile-assisted formation of C-C bonds is extremely small, and there is presently no definitive example of a biological Rauhut-Currier reaction. Biosynthesis of the polyketide insecticide spinosyn A in involves a [4 + 2]-cycloaddition and a subsequent intramolecular C-C bond formation catalyzed by SpnF and SpnL, respectively. Isotope tracer experiments and kinetic isotope effects, however, imply that the SpnL-catalyzed reaction proceeds without initial deprotonation of the substrate. The crystal structure of SpnL exhibits high similarity to SAM-dependent methyltransferases as well as SpnF. The residue Cys60 is also shown to reside in the SpnL active site, and the Cys60Ala SpnL mutant is found to be devoid of activity. Moreover, SpnL is covalently modified at Cys60 and irreversibly inactivated when it is coincubated with a fluorinated substrate analogue designed as a suicide inactivator of nucleophile-assisted C-C bond formation. These results suggest that SpnL catalyzes a biological Rauhut-Currier reaction.
已知能够催化亲核试剂辅助形成 C-C 键的酶的目录非常小，目前还没有生物学上 Rauhut-Currier 反应的明确例子。多酮杀虫剂 spinosyn A 的生物合成涉及[4+2]-环加成和随后的 SpnF 和 SpnL 分别催化的分子内 C-C 键形成。然而，同位素示踪实验和动力学同位素效应表明，SpnL 催化的反应在没有底物初始去质子化的情况下进行。SpnL 的晶体结构与 SAM 依赖性甲基转移酶以及 SpnF 具有高度相似性。残基 Cys60 也被证明位于 SpnL 的活性位点中，并且 Cys60Ala SpnL 突变体被发现没有活性。此外，SpnL 在 Cys60 处被共价修饰，并在与设计为亲核试剂辅助 C-C 键形成自杀失活剂的氟化底物类似物共孵育时不可逆失活。这些结果表明 SpnL 催化了一种生物学上的 Rauhut-Currier 反应。
