定向演化的新催化部位2-keto-3-deoxy-6-phosphogluconate醛缩酶的大肠杆菌。
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Wymer N,布坎南LV,亨德森D,梅塔N,堵塞CH, Pocivavsek L, Fierke CA, Toone EJ,奈史密斯JH
定向演化的新催化部位2-keto-3-deoxy-6-phosphogluconate醛缩酶的大肠杆菌。
结构。2001年1月10日,9 (1):1 - 9。
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11342129 (在PubMed]
- 文摘
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背景:醛缩酶是碳bond-forming酶一直被认定为有机化学家的有用的工具。然而,他们的效用有限部分窄底物的利用率。各种酶的定点诱变来改变他们的特异性已执行多年,通常没有预期的效果。最近定向进化一直雇佣工程师新活动到现有的脚手架。这种方法允许随机突变的基因,然后选择健身目的这些蛋白质所需的活动。迄今为止这种方法提供新颖的活动通过多种突变残留参与识别;在任何情况下都不关键催化残基被改变而活动保留。结果:我们报告一个双突变体的大肠杆菌2-keto-3-deoxy-6-phosphogluconate醛缩酶减少,但可衡量的酶活性和一个综合有用的衬底。突变被发现从定向进化实验。底物特异性的修改是通过改变活性部位的位置从一个β链赖氨酸相邻链而不是通过底物识别网站的修改。 The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution. CONCLUSIONS: These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.