Gliotoxin is a virulence factor of the human pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. The activity of this metabolite is mediated by a transannular disulfide bond, a hallmark of the epipolythiodiketopiperazine (ETP) family. Through the creation of fungal gene deletion mutants and heterologous protein expression, we unveiled the critical role of the cytochrome P450 monooxygenase (CYP450) GliC for the stepwise bishydroxylation of the diketopiperazine (DKP) core. We show for the first time the formation of the C–S bond from the DKP in a combined assay of GliC and the glutathione-S-transferase (GST) GliG in vitro. Furthermore, we present experimental evidence for an intermediary imine species. The flexible substrate scope of GliC and GliG in combination parallels P450/GST pairs used in eukaryotic phase I/II detoxification pathways.

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酶碳-硫键形成的重组揭示了真菌毒素生物合成中的排毒样策略

胶质毒素是人类病原体烟曲霉（Aspergillus fumigatus）的毒力因子，烟曲霉是侵袭性曲霉病的主要原因。这种代谢产物的活性是由跨环二硫键（表聚硫代二酮哌嗪（ETP）家族的标志）介导的。通过创建真菌基因缺失突变体和异源蛋白表达，我们揭示了细胞色素P450单加氧酶（CYP450）GliC对于二酮哌嗪（DKP）核的逐步双羟基化的关键作用。我们首次展示了在体外结合GliC和谷胱甘肽S-转移酶（GST）GliG的联合测定中从DKP形成C–S键的过程。此外，我们提出了一种中间亚胺物种的实验证据。GliC和GliG的柔性底物范围可与真核I / II排毒途径中使用的P450 / GST对组合使用。