Design principles for site-selective hydroxylation by a Rieske oxygenase.
Design principles for site-selective hydroxylation by a Rieske oxygenase.
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DOI：
10.1038/s41467-021-27822-3
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发表时间：
2022-01-11
影响因子：
16.6
通讯作者：
Bridwell-Rabb J
中科院分区：
文献类型：
--
作者：
Liu J;Tian J;Perry C;Lukowski AL;Doukov TI;Narayan ARH;Bridwell-Rabb J
Rieske加氧酶利用铁的反应性来进行具有化学挑战性的C-H键官能化反应。到目前为止，只有少数Rieske加氧酶的结构特征和非常少的信息存在关于这些酶如何使用一个共同的架构和一套的催化剂，以促进各种各样的反应。在此，我们详细介绍了两个Rieske加氧酶SxtT和GxtA如何使用不同的蛋白质区域来影响其催化的单羟基化反应的位点选择性。我们提出了高分辨率的晶体结构的SxtT和GxtA与本地β-石房蛤毒素醇和石房蛤毒素基板结合除了氙气加压结构的GxtA，揭示了一个基板的位置进入隧道的活性位点。最终，该结构信息允许鉴定分布在SxtT的三个区域之间的六个残基，其共同控制C-H羟基化事件的选择性。这些残基的取代产生SxtT变体，其完全适于表现出GxtA的非天然位点选择性和底物范围。重要的是，我们还发现，这些选择性区域是保守的，在其他结构特征的Rieske加氧酶，提供了一个框架，预测再利用和操纵Rieske加氧酶作为生物催化剂。SxtT和GxtA是参与麻痹性贝类毒素生物合成的Rieske加氧酶，并在毒素支架上的不同位置催化单羟基化反应。在这里，作者提出了SxtT和GxtA与天然底物β-石房蛤毒素醇和石房蛤毒素的晶体结构以及GxtA的氙加压结构，其揭示了通向活性位点的底物通道。通过基于结构的诱变研究，作者确定了三个不同蛋白质区域中的六个残基，这些残基决定了SxtT和GxtA的底物特异性和位点选择性。这些发现将有助于其他Rieske加氧酶的合理工程。
Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C–H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases have been structurally characterized and remarkably little information exists regarding how these enzymes use a common architecture and set of metallocenters to facilitate a diverse range of reactions. Herein, we detail how two Rieske oxygenases SxtT and GxtA use different protein regions to influence the site-selectivity of their catalyzed monohydroxylation reactions. We present high resolution crystal structures of SxtT and GxtA with the native β-saxitoxinol and saxitoxin substrates bound in addition to a Xenon-pressurized structure of GxtA that reveals the location of a substrate access tunnel to the active site. Ultimately, this structural information allowed for the identification of six residues distributed between three regions of SxtT that together control the selectivity of the C–H hydroxylation event. Substitution of these residues produces a SxtT variant that is fully adapted to exhibit the non-native site-selectivity and substrate scope of GxtA. Importantly, we also found that these selectivity regions are conserved in other structurally characterized Rieske oxygenases, providing a framework for predictively repurposing and manipulating Rieske oxygenases as biocatalysts. SxtT and GxtA are Rieske oxygenases that are involved in paralytic shellfish toxin biosynthesis and catalyze monohydroxylation reactions at different positions on the toxin scaffold. Here, the authors present crystal structures of SxtT and GxtA with the native substrates β-saxitoxinol and saxitoxin as well as a Xenon-pressurized structure of GxtA, which reveal a substrate access tunnel to the active site. Through structure-based mutagenesis studies the authors identify six residues in three different protein regions that determine the substrate specificity and site selectivity of SxtT and GxtA. These findings will aid the rational engineering of other Rieske oxygenases.
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影响因子：
5.6
作者：
Dumitru, Razvan;Jiang, Wen Zhi;Weeks, Donald P.;Wilson, Mark A.
通讯作者：
Wilson, Mark A.
DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
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
影响因子：
--
作者：
Ferraro, Daniel J.;Brown, Eric N.;Ramaswamy, S.
通讯作者：
Ramaswamy, S.
影响因子：
5.7
作者：
Ashikawa, Yuji;Fujimoto, Zui;Nojiri, Hideaki
通讯作者：
Nojiri, Hideaki