Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models.
Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models.
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DOI：
10.1038/nchem.2474
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发表时间：
2016-05
期刊：
影响因子：
21.8
通讯作者：
Arnold FH
中科院分区：
文献类型：
--
作者：
Dodani SC;Kiss G;Cahn JK;Su Y;Pande VS;Arnold FH
蛋白质结构元件的动态运动，特别是柔性环，与酶催化的各个方面密切相关。这些环区域的工程化可以改变蛋白质稳定性、底物结合，甚至显著影响酶功能。当这些灵活的区域在结构上无法解析时，结合大规模分子动力学模拟的计算重建可以用于指导工程策略。在这里，我们提出了一个合作的方法，包括实验和计算，导致发现的硝化细胞色素P450 TxtE的F/G环中的一个单一的突变，同时控制循环动力学和完全转移酶的区域选择性从C4到C5位置的L-色氨酸。此外，我们发现这种环突变天然存在于同源硝化P450的一个子集中，并证实这些未表征的酶专门产生5-硝基-L-色氨酸，这是一种以前未知的生物合成中间体。
The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding, and even dramatically impact enzyme function. When these flexible regions are structurally unresolvable, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here, we present a collaborative approach consisting of both experiment and computation that led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.