The design of artificial metalloenzymes is a challenging, yet ultimately highly rewarding objective because of the potential for accessing new-to-nature reactions. One of the main challenges is identifying catalytically active substrate–metal cofactor–host geometries. The advent of expanded genetic code methods for the in vivo incorporation of non-canonical metal-binding amino acids into proteins allow to address an important aspect of this challenge: the creation of a stable, well-defined metal-binding site. Here, we report a designed artificial metallohydratase, based on the transcriptional repressor lactococcal multidrug resistance regulator (LmrR), in which the non-canonical amino acid (2,2′-bipyridin-5yl)alanine is used to bind the catalytic Cu(II) ion. Starting from a set of empirical pre-conditions, a combination of cluster model calculations (QM), protein–ligand docking and molecular dynamics simulations was used to propose metallohydratase variants, that were experimentally verified. The agreement observed between the computationally predicted and experimentally observed catalysis results demonstrates the power of the artificial metalloenzyme design approach presented here.

中文翻译：

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包含非天然金属结合氨基酸的对映选择性人工金属水合酶的设计

人造金属酶的设计是一个具有挑战性，但最终具有很高回报的目标，因为它有可能获得从新到自然的反应。主要挑战之一是确定具有催化活性的底物-金属辅因子-主体几何形状。用于体内将非标准金属结合氨基酸掺入蛋白质的扩展遗传密码方法的出现，解决了这一挑战的一个重要方面：建立稳定，定义明确的金属结合位点。在这里，我们报道了一种基于转录阻遏物乳球菌多药抗性调节剂（LmrR）设计的人工金属水合酶，其中使用非规范性氨基酸（2,2'-联吡啶-5基）丙氨酸结合催化性Cu（II） 离子。从一组经验先决条件开始，结合了聚类模型计算（QM），蛋白质-配体对接和分子动力学模拟，提出了金属水合酶变体，并进行了实验验证。在计算预测和实验观察到的催化结果之间观察到的一致性证明了本文介绍的人工金属酶设计方法的强大功能。