Visualization of mutagenic nucleotide processing by Escherichia coli MutT, a Nudix hydrolase.
Visualization of mutagenic nucleotide processing by Escherichia coli MutT, a Nudix hydrolase.
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DOI：
10.1073/pnas.2203118119
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发表时间：
2022-05-24
影响因子：
11.1
通讯作者：
Yamagata, Yuriko
中科院分区：
文献类型：
--
作者：
Nakamura, Teruya;Yamagata, Yuriko
时间分辨x射线晶体学捕获了酶促反应中的瞬态元素，并对典型机制有了深入的了解。虽然Nudix水解酶通过水解活性参与细胞代谢，但其催化机制尚不明确。这是因为Nudix水解酶仅由一个高度保守的短基序定义，因为大多数机制是基于模拟反应状态的结构。MutT的时间分辨x射线晶体学跟踪了8-oxo-dGTP水解的过程，并使与三个金属离子顺序配位同步的水分子的亲核取代过程可视化。这些观察结果与先前的动力学分析一致，并表明了三金属离子机制。在Mg2+或Mn2+离子存在下，大肠杆菌MutT通过水解致突变的8-氧-2'-脱氧鸟苷5'-三磷酸（8-氧- dgtp）来防止突变。MutT是核苷二磷酸连接片段X （Nudix）水解酶超家族中研究最多的酶之一，广泛分布于生物体中。然而，大多数Nudix水解酶的催化机制，包括两个或三个金属离子的机制，仍然不清楚，因为这些机制是通过模拟反应状态的结构提出的，例如底物类似物配合物。在这里，我们使用生物底物8-oxo-dGTP和活性金属离子Mn2+，通过时间分辨x射线晶体学可视化了MutT的水解反应过程。该反应是通过将MutT晶体浸泡在MnCl2溶液中开始的，并通过在不同的时间点冻结晶体来停止。通过研究活性位点电子密度的时间过程和Mn2+离子的异常信号强度，共确定了5种中间结构。结构和电子密度表明，三个Mn2+离子结合到MutT的Nudix基序上，并对齐底物8-oxo-dGTP进行催化。伴随着三个Mn2+离子的配位，一个水分子与一个催化碱结合，在8-oxo-dGTP的β-磷上形成一个双核Mn2+中心进行亲核取代。使用Mg2+的反应条件也捕获了一个由3个Mg2+离子组成的络合物结构。本研究为了解Nudix水解酶的三金属离子机制提供了必要的结构细节，并提出一些Nudix水解酶具有这种机制。
Time-resolved X-ray crystallography has captured transient elements in enzymatic reactions and has advanced understanding regarding canonical mechanisms. Although Nudix hydrolases are involved in cellular metabolism via hydrolytic activities, the catalytic mechanisms remain elusive. This is because the Nudix hydrolases are defined only by a highly conserved short motif and because most mechanisms are based on the structures mimicking the reaction states. Time-resolved X-ray crystallography of MutT has followed the course of 8-oxo-dGTP hydrolysis and enabled visualization of the process of nucleophilic substitution by a water molecule that is synchronized with the sequential coordination of three metal ions. These observations are consistent with previous kinetic analysis and are indicative of a three-metal-ion mechanism. Escherichia coli MutT prevents mutations by hydrolyzing mutagenic 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) in the presence of Mg2+ or Mn2+ ions. MutT is one of the most studied enzymes in the nucleoside diphosphate-linked moiety X (Nudix) hydrolase superfamily, which is widely distributed in living organisms. However, the catalytic mechanisms of most Nudix hydrolases, including two- or three-metal-ion mechanisms, are still unclear because these mechanisms are proposed using the structures mimicking the reaction states, such as substrate analog complexes. Here, we visualized the hydrolytic reaction process of MutT by time-resolved X-ray crystallography using a biological substrate, 8-oxo-dGTP, and an active metal ion, Mn2+. The reaction was initiated by soaking MutT crystals in a MnCl2 solution and stopped by freezing the crystals at various time points. In total, five types of intermediate structures were refined by investigating the time course of the electron densities in the active site as well as the anomalous signal intensities of Mn2+ ions. The structures and electron densities show that three Mn2+ ions bind to the Nudix motif of MutT and align the substrate 8-oxo-dGTP for catalysis. Accompanied by the coordination of the three Mn2+ ions, a water molecule, bound to a catalytic base, forms a binuclear Mn2+ center for nucleophilic substitution at the β-phosphorus of 8-oxo-dGTP. The reaction condition using Mg2+ also captured a structure in complex with three Mg2+ ions. This study provides the structural details essential for understanding the three-metal-ion mechanism of Nudix hydrolases and proposes that some of the Nudix hydrolases share this mechanism.
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发现聚合酶诱导的氧化核苷酸的细胞毒性。
影响因子：
64.8
作者：
Freudenthal, Bret D.;Beard, William A.;Perera, Lalith;Shock, David D.;Kim, Taejin;Schlick, Tamar;Wilson, Samuel H.
通讯作者：
Wilson, Samuel H.
DOI：
10.1073/pnas.2116159118
发表时间：
2022-01-04
影响因子：
11.1
作者：
Choi WS;Weng PJ;Yang W
通讯作者：
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影响因子：
2.9
作者：
Ito, R;Hayakawa, H;Ishibashi, T
通讯作者：
Ishibashi, T
影响因子：
2.9
作者：
ABEYGUNAWARDANA, C;WEBER, DJ;MILDVAN, AS
通讯作者：
MILDVAN, AS
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K