Catalytic mechanism and molecular engineering of quinolone biosynthesis in dioxygenase AsqJ.
Catalytic mechanism and molecular engineering of quinolone biosynthesis in dioxygenase AsqJ.
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DOI：
10.1038/s41467-018-03442-2
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发表时间：
2018-03-21
影响因子：
16.6
通讯作者：
Kaila VRI
中科院分区：
文献类型：
--
作者：
Mader SL;Bräuer A;Groll M;Kaila VRI
最近发现的依赖FeII/α-酮戊二酸的双加氧酶AsqJ可以立体选择性地催化喹诺酮类生物碱的多步合成，这些天然产物具有重要的生物医学应用。为了探索这一难以捉摸的催化过程的分子机制，我们将多尺度量子和经典分子模拟与X射线结晶学和体外生化活性研究相结合。我们发现底物的甲基化对于AsqJ的活性是必不可少的，建立了微调活性部位内π堆积相互作用的分子菌株。为了合理设计修饰底物的AsqJ，我们放大了活性部位内的弥散相互作用。我们证明了工程酶对非甲基化替代物的催化活性大大增强，证实了我们的计算数据，并在1.55 á分辨率下解析了高分辨率X射线结构。我们的联合发现提供了对AsqJ功能的关键机制理解，并展示了计算和实验数据的结合如何能够合理地设计酶。双加氧酶AsqJ的催化活性严格依赖于喹诺酮类底物的甲基化。在这里，作者应用分子模拟、X射线结晶学和体外生物化学研究来设计双加氧酶AsqJ，以提高对修饰的非甲基化代谢物的催化活性。
The recently discovered FeII/α-ketoglutarate-dependent dioxygenase AsqJ from Aspergillus nidulans stereoselectively catalyzes a multistep synthesis of quinolone alkaloids, natural products with significant biomedical applications. To probe molecular mechanisms of this elusive catalytic process, we combine here multi-scale quantum and classical molecular simulations with X-ray crystallography, and in vitro biochemical activity studies. We discover that methylation of the substrate is essential for the activity of AsqJ, establishing molecular strain that fine-tunes π-stacking interactions within the active site. To rationally engineer AsqJ for modified substrates, we amplify dispersive interactions within the active site. We demonstrate that the engineered enzyme has a drastically enhanced catalytic activity for non-methylated surrogates, confirming our computational data and resolved high-resolution X-ray structures at 1.55 Å resolution. Our combined findings provide crucial mechanistic understanding of the function of AsqJ and showcase how combination of computational and experimental data enables to rationally engineer enzymes. The catalytic activity of dioxygenase AsqJ is strictly relying on the methylation of quinolone substrates. Here, the authors apply molecular simulations, X-ray crystallography and in vitro biochemical studies to the engineering of dioxygenase AsqJ with improved catalytic activity for modified non-methylated surrogates.
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影响因子：
5.5
作者：
Contreras-Garcia, Julia;Johnson, Erin R.;Keinan, Shahar;Chaudret, Robin;Piquemal, Jean-Philip;Beratan, David N.;Yang, Weitao
通讯作者：
Yang, Weitao
DOI：
10.1039/p29930000799
发表时间：
1993-05-01
期刊：
JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2
影响因子：
--
作者：
KLAMT, A;SCHUURMANN, G
通讯作者：
SCHUURMANN, G
影响因子：
5.1
作者：
An, Chun-Yan;Li, Xiao-Ming;Wang, Bin-Gui
通讯作者：
Wang, Bin-Gui
影响因子：
5.5
作者：
Best, Robert B.;Zhu, Xiao;Shim, Jihyun;Lopes, Pedro E. M.;Mittal, Jeetain;Feig, Michael;MacKerell, Alexander D., Jr.
通讯作者：
MacKerell, Alexander D., Jr.
影响因子：
4.4
作者：
BECKE, AD
通讯作者：
BECKE, AD