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 Saccharopolyspora spinosa 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.

中文翻译：

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多杀菌素 A 生物合成过程中酶催化的 Rauhut-Currier 反应的证据

已知催化亲核试剂辅助形成 C-C 键的酶目录非常少，目前还没有生物 Rauhut-Currier 反应的明确例子。多刺糖多孢菌中聚酮类杀虫剂多杀菌素 A 的生物合成涉及分别由 SpnF 和 SpnL 催化的 [4 + 2]-环加成和随后的分子内 C-C 键形成。然而，同位素示踪实验和动力学同位素效应暗示 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 也显示存在于 SpnL 活性位点，并且发现 Cys60Ala SpnL 突变体没有活性。此外，SpnL 在 Cys60 处被共价修饰，并且当它与氟化底物类似物共温育时，它被设计为亲核试剂辅助 C-C 键形成的自杀灭活剂。这些结果表明 SpnL 催化了生物 Rauhut-Currier 反应。