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首页> 外文期刊>Journal of Molecular Biology >The transition of human estrogen sulfotransferase from generalist to specialist using directed enzyme evolution
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The transition of human estrogen sulfotransferase from generalist to specialist using directed enzyme evolution

机译：人类雌激素磺基转移酶使用定向酶进化从通才向专家过渡

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摘要

Broad specificity is believed to be a property of primordial enzymes that diverged during natural protein evolution to produce highly specific and efficient enzymes. Human estrogen sulfotransferase (SULT1E1) is a broad-specificity enzyme that detoxifies a variety of chemicals, including estrogens, by the transfer of sulfate. To study the molecular basis for the broad specificity of this enzyme and to investigate the process of SULT1E1 specialization, we have adopted a directed enzyme evolution approach. Using two iterative rounds of evolution, we generated SULT1E1 mutants with increased thermostability and narrower specificity from the broadly specific wild-type enzyme. To identify mutants with enhanced specificity, we developed an unbiased screening assay to assess sulfate transfer to three different acceptors in parallel. Such an assay enabled the isolation of SULT1E1 mutants with enhanced or wild-type activity toward an estrogen acceptor and significantly reduced activity for phenol or coumarin type of acceptors, leading to up to 3 orders of magnitude increase in specificity. We found that mutations conferring novel specificity are located in the vicinity of the active site and thus may play a direct role in reshaping the acceptor-binding site. Finally, such mutations resulted in reduced SULT1E1 thermostability, revealing a trade-off between SULT1E1 thermostability and acquisition of novel function.
机译：广泛的特异性被认为是原始酶的一种特性，该酶在天然蛋白质进化过程中发生分化，从而产生高度特异性和高效的酶。人雌激素磺基转移酶（SULT1E1）是一种广泛特异性的酶，通过转移硫酸盐使多种化学物质（包括雌激素）解毒。为了研究这种酶的广泛特异性的分子基础，并研究SULT1E1专业化的过程，我们采用了定向酶进化方法。使用两个迭代的进化轮，我们从广泛的野生型酶中产生了具有更高热稳定性和更窄特异性的SULT1E1突变体。为了鉴定具有增强的特异性的突变体，我们开发了一种无偏筛选试验，以评估硫酸盐平行转移至三个不同受体的转移。这种测定法能够分离出对雌激素受体具有增强或野生型活性，对苯酚或香豆素类型的受体具有明显降低的活性的SULT1E1突变体，从而导致特异性提高多达3个数量级。我们发现赋予新颖特异性的突变位于活性位点附近，因此可能在重塑受体结合位点中起直接作用。最后，此类突变导致SULT1E1热稳定性降低，从而揭示了SULT1E1热稳定性与获得新功能之间的权衡。

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