Genetic Incorporation of Selenotyrosine Significantly Improves Enzymatic Activity of Agrobacterium radiobacter Phosphotriesterase

Tyrosine plays important roles in many enzymes. To facilitate enzyme design, mechanistic studies and minimize structural perturbation in the active site, here we report the genetic incorporation of a novel unnatural amino acid selenotyrosine (SeHF), which has single-atom replacement in comparison to tyrosine. The arPTE-(Agrobacterium radiobacter Phosphotriesterase) Tyr309SeHF mutant exhibits a significant 12-fold increase in k(cat) and 3.2-fold enhancement in k(cat)/K-M at pH 7.0. Molecular dynamics simulations show that the SeHF309 mutation results in a conformational switch which opens up the product release pocket and increases the product release rate, thereby elevating the overall enzyme activity. Significant improvement of the catalytic efficiency at neutral pH by single unnatural amino acid (UAA) mutation broadens the application of this enzyme, and provides valuable insights to the mechanism. Our method represents a new approach for designing enzymes with enhanced activity.

酪氨酸在许多酶中发挥重要作用。为了促进酶的设计、机理研究以及尽量减少活性位点的结构扰动，在此我们报道了一种新型非天然氨基酸硒代酪氨酸（SeHF）的基因掺入，与酪氨酸相比，它具有单原子替换。农杆菌磷酸三酯酶（arPTE）的Tyr309SeHF突变体在pH 7.0时，$k_{cat}$显著增加12倍，$k_{cat}/K_M$提高3.2倍。分子动力学模拟表明，SeHF309突变导致一种构象转变，打开了产物释放口袋并提高了产物释放速率，从而提高了整体酶活性。通过单个非天然氨基酸（UAA）突变在中性pH下催化效率的显著提高拓宽了这种酶的应用，并为其作用机制提供了有价值的见解。我们的方法代表了一种设计具有增强活性的酶的新途径。