Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate
Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate
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DOI：
10.1002/anie.201606722
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发表时间：
2016-11-07
影响因子：
16.6
通讯作者：
Ang, Ee Lui
中科院分区：
文献类型：
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作者：
Sun, Huihua;Yeo, Wan Lin;Ang, Ee Lui
氟化酶为温和条件下的选择性氟化提供了一种环境友好的替代方法。然而，它们的多样性在自然界中是有限的，它们还有待于通过定向进化来实现。在这里，我们报道了氟化酶FlA1的定向进化，以促进非天然底物5‘-氯-5’-脱氧腺苷(5‘-ClDA)转化为5’-氟-5‘-脱氧腺苷(5’-FDA)。进化的突变体fah2081(A279Y)和fah2114(F213Y，A279L)成功地应用于5-‘[F-18]FDA的放射合成，总的放射化学转化率(RCC)比野生型FlA1高3倍以上。两步反应的动力学研究表明，在5‘-ClDA转化为沙腺苷-L-蛋氨酸(SAM)的过程中，变异体的k(CAT)值显著提高，而在SAM转化为5’-FDA的过程中，k(CAT)值显著降低。
Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non-native substrate 5'-chloro-5'-deoxyadenosine (5'-ClDA) into 5'-fluoro-5'-deoxyadenosine (5'-FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5-'[F-18] FDA, with overall radiochemical conversion (RCC) more than 3-fold higher than wild-type FlA1. Kinetic studies of the two-step reaction revealed that the variants show a significantly improved k(cat) value in the conversion of 5'-ClDA into Sadenosyl-l-methionine (SAM) but a reduced k(cat) value in the conversion of SAM into 5'-FDA.