Cherepanov Alexey V, Doroshenko Elena V, Matysik Jörg, de Vries Simon, de Groot Huub J M
Biophysical Organic Chemistry/Solid-State NMR, Leiden Institute of Chemistry, Faculty of Mathematics and Natural Sciences, Leiden University, Einsteinweg 55, 2333CC Leiden, The Netherlands.
Proc Natl Acad Sci U S A. 2008 Jun 24;105(25):8563-8. doi: 10.1073/pnas.0709140105. Epub 2008 Jun 18.
DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD(+) and assistance of a divalent metal cofactor Mg(2+). Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover (31)P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase-nucleotide complex is triggered in situ by the photo release of caged Mg(2+), and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4-5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations.
DNA连接酶利用ATP或NAD(+)中磷酸酐键的化学能以及二价金属辅助因子Mg(2+)的协助来封闭双链DNA中的缺口。连接酶催化的分子细节对于理解活细胞中金属促进的磷酰基转移反应机制至关重要，这些反应负责广泛的过程，例如DNA复制和转录、信号传导和分化、能量耦合和代谢。在此，我们报道了噬菌体T4 DNA连接酶催化的腺苷酰转移的单周转(31)P固态核磁共振研究。通过笼形Mg(2+)的光释放原位触发高能共价连接酶-核苷酸复合物的形成，并通过核磁共振监测依次形成的中间体。反应动力学和化学位移变化分析表明，反应4-5小时后，五配位磷烷中间体积累至总反应物种的35%。这是DNA连接酶催化的腺苷酰转移的缔合性质的直接实验证据。旋转固体中的核磁共振光谱被引入作为记录反应过程分子动态影像的分析工具。目前的工作开创了生物化学转化结构研究的一个有前景的方向。
