Fachbereich Chemie, Abteilung Biochemie, Technische Universität Kaiserslautern, D-67663, Kaiserslautern, Germany.
Institut für Biochemie, Zentrum für Human- und Molekularbiologie (ZHMB), Universität des Saarlandes, D-66123, Saarbrücken, Germany.
Nat Commun. 2020 Apr 7;11(1):1725. doi: 10.1038/s41467-020-15441-3.
Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.
I 类谷氧还蛋白是具有酶活性的、依赖谷胱甘肽的氧化还原酶，而 II 类谷氧还蛋白通常是无酶活性的、Fe-S 簇结合蛋白。具有酶活性的谷氧还蛋白既具有与谷胱甘肽化二硫键底物反应的谷胱甘肽支架位点，又具有与还原型谷胱甘肽反应的谷胱甘肽激活位点。在这里，我们使用酵母 ScGrx7 作为模型蛋白，全面鉴定和表征了来自四个不同蛋白区域的关键残基以及共价结合的谷胱甘肽部分，并定量它们对两个相互作用位点的贡献。此外，我们开发了一种基于还原敏感 GFP2 的测定法，可实时评估活细胞内谷氧还蛋白的结构-功能关系。最后，我们利用该测定法快速筛选多种谷氧还蛋白突变体，最终使我们能够将具有酶活性和无酶活性的谷氧还蛋白相互转化。总之，我们全面了解了谷氧还蛋白催化的机制基础，并阐明了两类主要谷氧还蛋白之间的决定结构差异。
