Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstrasse 11a, 07745, Jena, Germany.
Technical University of Munich, Center for Protein Assemblies, Ernst-Otto-Fischer-Strasse 8, 85747, Garching, Germany.
Angew Chem Int Ed Engl. 2021 Jun 14;60(25):14188-14194. doi: 10.1002/anie.202104372. Epub 2021 May 14.
Glutathione-S-transferases (GSTs) usually detoxify xenobiotics. The human pathogenic fungus Aspergillus fumigatus however uses the exceptional GST GliG to incorporate two sulfur atoms into its virulence factor gliotoxin. Because these sulfurs are essential for biological activity, glutathionylation is a key step of gliotoxin biosynthesis. Yet, the mechanism of carbon-sulfur linkage formation from a bis-hydroxylated precursor is unresolved. Here, we report structures of GliG with glutathione (GSH) and its reaction product cyclo[-l-Phe-l-Ser]-bis-glutathione, which has been purified from a genetically modified A. fumigatus strain. The structures argue for stepwise processing of first the Phe and second the Ser moiety. Enzyme-mediated dehydration of the substrate activates GSH and a helix dipole stabilizes the resulting anion via a water molecule for the nucleophilic attack. Activity assays with mutants validate the interactions of GliG with the ligands and enrich our knowledge about enzymatic C-S bond formation in gliotoxin and epipolythiodioxopiperazine (ETP) natural compounds in general.
谷胱甘肽-S-转移酶（GSTs）通常可以解毒异源物。然而，人类致病性真菌烟曲霉利用特殊的 GST GliG 将两个硫原子掺入其毒力因子Gliotoxin 中。由于这些硫原子对于生物活性是必需的，因此谷胱甘肽化是Gliotoxin 生物合成的关键步骤。然而，从双羟基化前体形成碳-硫键的机制仍未解决。在这里，我们报道了GliG 与谷胱甘肽（GSH）及其反应产物环[-l-Phe-l-Ser]-双谷胱甘肽的结构，该结构已从遗传修饰的烟曲霉菌株中纯化出来。这些结构表明，首先是苯丙氨酸，然后是丝氨酸部分的逐步加工。酶介导的底物脱水使 GSH 活化，螺旋偶极通过水分子稳定生成的阴离子，以便进行亲核攻击。用突变体进行的活性测定验证了 GliG 与配体的相互作用，并丰富了我们对 Gliotoxin 和一般的 epipolythiodioxopiperazine (ETP) 天然化合物中酶促 C-S 键形成的认识。
