Hydrophilicity-Based Engineering of the Active Pocket of D-Amino Acid Oxidase Leading to Highly Improved Specificity toward D-Glufosinate.
Hydrophilicity-Based Engineering of the Active Pocket of D-Amino Acid Oxidase Leading to Highly Improved Specificity toward D-Glufosinate.
复制标题
D-氨基酸氧化酶活性袋的基于亲水性的工程可显着提高对 D-草铵膦的特异性。
DOI：
10.1002/anie.202212720
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发表时间：
2022
影响因子：
--
通讯作者：
W. Zeng
中科院分区：
文献类型：
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作者：
K. Yang;Bin Huang;Charles Amanze;Zhen Yan;Guanzhou Qiu;Xue;Hong;W. Zeng
由于其严格的立体特异性，D-氨基酸氧化酶（DAAO）使得合成L-氨基酸变得非常容易。然而，野生型酶对非天然底物如D-草铵膦（D-PPT）的低活性限制了其应用。在此，从纤细红酵母DAAO（Rg DAAO）直接进化使用一个突变体取代饱和诱变策略，产生一个突变体具有显着增加的催化活性对D-PPT。与许多WT-DAAO相比，该突变体对亲水性底物显示出独特的催化特性。同源模建和分子动力学模拟分析表明，反应袋的延伸和更大的亲水性是活性增强的原因。本研究建立了一条高效合成除草剂L-PPT的酶促合成路线，为非天然氨基酸生物合成的酶工程研究提供了新的思路。
Due to its stringent stereospecificity, D-amino acid oxidase (DAAO) has made it incredibly easy to synthesize L-amino acids. However, the wild-type enzyme's low activity toward unnatural substrates like D-glufosinate (D-PPT) restricts its application. Herein, DAAO from Rhodotorula gracilis ( Rg DAAO) was directly evolved using a hydrophilicity-substitution saturation mutagenesis strategy, yielding a mutant with significantly increased catalytic activity against D-PPT. The mutant displays distinctive catalytic properties to hydrophilic substrates compared to numerous WT-DAAOs. The analysis of homology modeling and molecular dynamic simulation demonstrate that the extended reaction pocket with greater hydrophilicity was the reason for the enhanced activity. The current study established an enzymatic synthetic route to L-PPT, an excellent herbicide, with high efficiency, and the proposed strategy provided a new viewpoint on enzyme engineering for the biosynthesis of unnatural amino acids.