Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO₂ as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin‐mediated drug‐delivery of RGD‐modified DLDH (DLDH^RGD), leading to apoptotic cell death. In these experiments, DLDH^RGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA‐binding capability of this enzyme. To explore this interaction under cell‐free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel‐shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA‐binding. A Docking algorithm for prediction of protein‐DNA complexes, Paradoc, identified a putative DNA binding site at the C‐terminus of DLDH. Our finding that TiO₂‐bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.

中文翻译：

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二氢硫辛酰胺脱氢酶兼职作为 DNA 螯合剂。


二氢硫辛酰胺脱氢酶 (DLDH) 是一种线粒体酶，是丙酮酸脱氢酶复合物的重要组成部分。一系列证据表明，许多脱氢酶除了氧化还原酶活性外，还具有不相关的作用，称为兼职作用。作为这些活动的一部分，我们已经证明 DLDH 与 TiO ₂结合并产生活性氧 (ROS)。这种 ROS 产生能力通过整合素介导的 RGD 修饰的 DLDH (DLDH ^RGD ) 药物输送用于癌症治疗，导致细胞凋亡。在这些实验中，DLDH ^RGD不仅在细胞质中积累，而且迁移到细胞核，表明该酶具有潜在的 DNA 结合能力。为了探索无细胞条件下的这种相互作用，我们通过凝胶位移测定和分析超速离心分析了 DLDH 与噬菌体 lambda (λ) DNA 的结合，显示两者之间形成了复合物，从而导致 DLDH 分子完全覆盖 DNA 分子。 DNA 结合不影响 DLDH 酶活性，表明 DLDH 在 DNA 结合时既没有构象变化，也没有活性位点阻碍。用于预测蛋白质-DNA 复合物的对接算法 Paradoc 确定了 DLDH C 末端的假定 DNA 结合位点。我们发现 TiO ₂结合的 DLDH 未能与 DNA 形成复合物，这表明两个位点之间存在部分重叠。总之，DLDH 与 DNA 的结合呈现出一种新颖的月光活性，可用于癌症治疗中的 DNA 烷基化。