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观察一价金属离子依赖性和组氨酸促进的 His-Me 家族 I-PpoI 核酸内切酶催化作用。

Observing one-divalent-metal-ion-dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis .

机构信息

Department of Biosciences, Rice University, Houston, United States.

出版信息

Elife. 2024 Aug 14;13:RP99960. doi: 10.7554/eLife.99960.

DOI:10.7554/eLife.99960
PMID:39141555
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11325842/
Abstract

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.

摘要

金属离子依赖的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的 DNA 水解和合成的催化细节，揭示了多个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和需要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，已被用于生物技术和生物医学应用。然而，目前尚不清楚 His-Me 核酸酶中的单个金属离子与组氨酸一起如何促进水的去质子化、亲核攻击和磷酸二酯键的断裂。通过观察 His-Me I-PpoI 核酸酶作为模型系统的 DNA 水解，我们证明在其催化过程中只需要一个二价金属离子。此外，我们还揭示了亲核水可能的几种去质子化途径。有趣的是，在催化过程中，单金属离子的结合和水的去质子化是协同进行的。我们的研究结果揭示了 His-Me 核酸酶的催化细节，这与多金属离子依赖的 DNA 聚合酶和核酸酶不同。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/cb93d7a3df22/elife-99960-fig6-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/c3191f061b03/elife-99960-fig1.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/c38be3aa2f6b/elife-99960-fig2.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/3789151f89a4/elife-99960-fig3-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/0f0c869baec8/elife-99960-fig3-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/3c37fb398867/elife-99960-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/3fc66210b1ed/elife-99960-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/b3bae3eab57d/elife-99960-fig5-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/56fb06bc39c3/elife-99960-fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/cb93d7a3df22/elife-99960-fig6-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/c3191f061b03/elife-99960-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/aa6bcd5f96ba/elife-99960-fig1-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/6c1af1e59b44/elife-99960-fig1-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/d5d9444cc5e5/elife-99960-fig1-figsupp3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/c38be3aa2f6b/elife-99960-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/71d750dfb3d2/elife-99960-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/3789151f89a4/elife-99960-fig3-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/0f0c869baec8/elife-99960-fig3-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/3c37fb398867/elife-99960-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/3fc66210b1ed/elife-99960-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/b3bae3eab57d/elife-99960-fig5-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/56fb06bc39c3/elife-99960-fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b28/11325842/cb93d7a3df22/elife-99960-fig6-figsupp1.jpg

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