Construction of ATP-Switched Allosteric Antioxidant Selenoenzyme

Rational redesign of allosteric protein offers an efficient strategy to develop switchable biocatalysts. By combining the computational design and protein engineering, a glutathione peroxidase (GPx)-like active center that contains the catalytic selenocysteine (Sec) residue and substrate-binding Arg residue was precisely incorporated into the allosteric domain of adenylate kinase (AKe). The engineered selenoenzyme shows not only high GPx activity but also adenosine triphosphate (ATP)-responsive catalytic property, which is regulated by its opened to closed conformational change upon ATP binding. Theoretical and mutational analysis reveals that the synergistic effect of electrostatic interactions and van der Waals (vdW) interactions for substrate recognition is a major contribution to the high activity. The mitochondrial oxidative damage experiment further demonstrated its antioxidant ability at the subcellular level, offering a potential application toward controllable catalysis in vivo.

变构蛋白的合理重新设计为开发可切换生物催化剂提供了有效的策略。通过将计算设计和蛋白质工程相结合，包含催化硒代半胱氨酸（Sec）残基和底物结合精氨酸残基的谷胱甘肽过氧化物酶（GPx）样活性中心被精确地整合到腺苷酸激酶（AKe）的变构结构域中。工程化的硒酶不仅表现出高 GPx 活性，而且还具有三磷酸腺苷 (ATP) 响应催化特性，这是通过 ATP 结合时其从打开到关闭的构象变化来调节的。理论和突变分析表明，静电相互作用和范德华（vdW）相互作用对底物识别的协同效应是高活性的主要贡献。线粒体氧化损伤实验进一步证明了其在亚细胞水平上的抗氧化能力，为体内可控催化提供了潜在的应用。