The non-ribosomal peptide gliotoxin, which autoinduces its own biosynthesis, has potent anti-fungal activity, especially in the combined absence of the gliotoxin oxidoreductase GliT and bis-thiomethyltransferase GtmA. Dithiol gliotoxin (DTG) is a substrate for both of these enzymes. Herein we demonstrate that DTG chelates Zn²⁺ (m/z 424.94), rapidly chelates Zn²⁺ from Zn(4-(2-pyridylazo)-resorcinol) (Zn(PAR)₂) and also inhibits a Zn²⁺-dependent alkaline phosphatase (AP). Zn²⁺ addition rescues AP function following DTG-associated inhibition, and pre-incubation of DTG with Zn²⁺ completely protects AP activity. Zn²⁺ (1–50 μM) also significantly relieves the potent gliotoxin-mediated inhibition of Aspergillus fumigatus ΔgliT::ΔgtmA (p < 0.05), which infers in vivo dithiol gliotoxin-mediated sequestration of free Zn²⁺ or chelation from intracellular metalloenzymes as inhibitory mechanisms. Quantitative proteomic analysis revealed that excess Zn²⁺ alters the effect of gliotoxin on A. fumigatus ΔgliT, with differential abundance of secondary metabolism-associated proteins in the combinatorial condition. GtmA abundance increased 18.8 fold upon co-addition of gliotoxin and Zn²⁺ compared to gliotoxin alone, possibly to compensate for disruption to GtmA activity, as seen in in vitro assays. Furthermore, DTG effected significant in vitro aggregation of a number of protein classes, including Zn²⁺-dependent enzymes, while proteins were protected from aggregation by pre-incubating DTG with Zn²⁺. We conclude that DTG can act in vivo as a Zn²⁺ chelator, which can significantly impede A. fumigatus growth in the absence of GliT and GtmA.

中文翻译：

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烟曲霉 中表聚硫代二氧杂哌嗪二硫醇gliotoxin对锌螯合的系统影响：天然产品功能的新方向†

自动诱导自身生物合成的非核糖体肽胶体毒素具有有效的抗真菌活性，尤其是在胶体毒素氧化还原酶GliT和双硫代甲基转移酶GtmA不存在的情况下尤其如此。二硫醇gliotoxin（DTG）是这两种酶的底物。在本文中，我们证明DTG螯合Zn ²⁺（m / z 424.94），从Zn（4-（2-吡啶基偶氮）-间苯二酚）（Zn（PAR）₂）快速螯合Zn ²⁺，并且还抑制Zn ²⁺依赖性碱性磷酸酶（AP）。锌^2+的添加可在DTG相关抑制后恢复AP功能，并且将TG 2与Zn ²⁺一起预孵育可完全保护AP活性。锌²⁺（1-50μM）也显著减轻的有效的胶霉毒素介导的抑制烟曲霉Δ GLIT ::Δ GTMA（p <0.05），其推断在体内二硫醇胶霉毒素介导的不含Zn螯合²⁺或螯合细胞内金属酶作为抑制机制。定量蛋白质组分析表明，过量的Zn ²⁺涂改胶霉毒素的效果上烟曲霉Δ GLIT，在组合条件差动丰度次级代谢相关的蛋白质。gliotoxin和Zn ²⁺共添加后，GtmA丰度增加了18.8倍如体外实验中所见，与单独使用的gliotoxin相比，可能是为了补偿对GtmA活性的破坏。此外，DTG对包括Zn ²⁺依赖的酶在内的许多蛋白质类别均具有显着的体外聚集作用，而DTG与Zn ^2+的预孵育可保护蛋白质免受聚集作用。我们得出结论，DTG可以在体内充当Zn ²⁺螯合剂，在没有GliT和GtmA的情况下，它可以显着阻碍烟曲霉的生长。