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首页> 美国卫生研究院文献>Protein Science : A Publication of the Protein Society >Free energy calculations elucidate substrate binding gating mechanism and tolerance‐promoting mutations in herbicide target 4‐hydroxyphenylpyruvate dioxygenase
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Free energy calculations elucidate substrate binding gating mechanism and tolerance‐promoting mutations in herbicide target 4‐hydroxyphenylpyruvate dioxygenase

机译：自由能计算阐明了除草剂靶标4-羟苯基丙酮酸双加氧酶的底物结合门控机制和促进耐受的突变

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摘要

4‐Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the second reaction in the tyrosine catabolism and is linked to the production of cofactors plastoquinone and tocopherol in plants. This important biological role has put HPPD in the focus of current herbicide design efforts including the development of herbicide‐tolerant mutants. However, the molecular mechanisms of substrate binding and herbicide tolerance have yet to be elucidated. In this work, we performed molecular dynamics simulations and free energy calculations to characterize active site gating by the C‐terminal helix H11 in HPPD. We compared gating equilibria in ( ) and ( ) wild‐type proteins retrieving the experimentally observed preferred orientations from the simulations. We investigated the influence of substrate and product binding on the open–closed transition and discovered a ligand‐mediated conformational switch in H11 that mediates rapid substrate access followed by active site closing and efficient product release through H11 opening. We further studied H11 gating in mutant HPPD, and found large differences with correlation to experimentally measured herbicide tolerance. The computational findings were then used to design a new mutant HPPD protein that showed increased tolerance to six commercially available HPPD inhibitors in biochemical in vitro experiments. Our results underline the importance of protein flexibility and conformational transitions in substrate recognition and enzyme inhibition by herbicides.
机译：4-羟基苯丙酮酸双加氧酶（HPPD）催化酪氨酸分解代谢中的第二个反应，并与植物中辅因子质体醌和生育酚的产生有关。这种重要的生物学作用使HPPD成为当前除草剂设计工作的重点，包括开发耐除草剂的突变体。但是，底物结合和除草剂耐受性的分子机制尚未阐明。在这项工作中，我们进行了分子动力学模拟和自由能计算，以表征HPPD中C末端螺旋H11的活性位点门控。我们比较了（）和（）野生型蛋白质中的门控平衡，从模拟中检索了实验观察到的优选方向。我们研究了底物和产物结合对开闭过渡的影响，并发现了H11中的一个由配体介导的构象转换，该开关介导了快速的底物进入，随后通过H11的开放进行了主动位点封闭和有效的产品释放。我们进一步研究了突变型HPPD中的H11门控，发现与实验测得的除草剂耐受性相关的差异很大。计算结果随后被用于设计新的突变HPPD蛋白，该蛋白在生化体外实验中显示出对六种市售HPPD抑制剂的增加的耐受性。我们的结果强调了在基质识别和除草剂酶抑制中蛋白质柔韧性和构象转变的重要性。

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