Chang Caleb, Zhou Grace, Gao Yang
Department of Biosciences, Rice University, Houston, Texas, 77005, USA.
bioRxiv. 2024 Jul 11:2024.05.02.592236. doi: 10.1101/2024.05.02.592236.
Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The histidine-metal (His-Me) superfamily nucleases are renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how the single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.
金属离子依赖性核酸酶在细胞防御和生物技术应用中发挥着关键作用。时间分辨晶体学解析了金属离子依赖性DNA水解和合成的催化细节，揭示了多种金属离子在催化过程中的重要作用。组氨酸-金属（His-Me）超家族核酸酶以结合一个二价金属离子并需要一个保守的组氨酸来促进催化而闻名。许多His-Me家族核酸酶，包括归巢内切核酸酶和Cas9核酸酶，已被应用于生物技术和生物医学领域。然而，目前尚不清楚His-Me核酸酶中的单个金属离子如何与组氨酸一起促进水的去质子化、亲核攻击和磷酸二酯键断裂。通过以His-Me I-PpoI核酸酶为模型系统观察DNA水解，我们证明其催化过程中仅需要一个二价金属离子。此外，我们发现了亲核水的几种可能的去质子化途径。有趣的是，在催化过程中，单个金属离子的结合和水的去质子化是协同进行的。我们的结果揭示了His-Me核酸酶的催化细节，这与多金属离子依赖性DNA聚合酶和核酸酶不同。
