Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0356, USA.
Science. 2014 Dec 19;346(6216):1525-8. doi: 10.1126/science.1259680.
The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(k(cat)/K(m))/k(uncat)] for ampicillin hydrolysis of 2.3 × 10(6) and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.
新酶活性的产生主要依赖于重新利用预先存在的蛋白质折叠内部结构，因为从第一原理设计具有功能的三维蛋白质结构具有挑战性。在这里，我们报告了一种人工金属β-内酰胺酶，它是通过将结构和功能上不相关的单体氧化还原蛋白自组装成具有界面催化锌位的四聚体组装体而构建的。设计的金属β-内酰胺酶在大肠杆菌周质中具有功能，并使细菌能够在氨苄青霉素处理后存活。文库的体内筛选产生了一种变体，其氨苄青霉素水解的催化效率[(k(cat)/K(m))/k(uncat)]为 2.3×10(6)，并且具有靠近活性位点的高度可移动环的出现，这是天然β-内酰胺酶的关键组件，使其能够进行底物相互作用。
