Design and evolution of new catalytic activity with an existing protein scaffold
Design and evolution of new catalytic activity with an existing protein scaffold
复制标题
DOI：
10.1126/science.1118953
复制
发表时间：
2006-01-27
期刊：
影响因子：
56.9
通讯作者：
Kim, HS
中科院分区：
文献类型：
--
作者：
Park, HS;Nam, SH;Kim, HS
设计具有新功能和性质的酶一直是蛋白质工程的目标。在这里，我们报告了一种改变现有蛋白质支架催化活性的策略。这是通过插入、缺失和取代几个活性位点环同时掺入和调节功能元件，然后进行点突变以微调活性来实现的。使用这种方法，我们能够将β-内酰胺酶活性引入到glycoprotein酶II的α β/β α金属水解酶支架中。所得的酶evMBL 8（进化的金属β-内酰胺酶8）完全失去了其原始活性，而是催化头孢噻肟的水解，（k（cat）/K-m）（app）为1.8 x 10（2）（摩尔/滴度）（-1）秒（-1），从而将头孢噻肟对大肠杆菌生长的抗性提高了约100倍。
The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce beta-lactamase activity into the alpha beta/beta alpha metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo beta-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (k(cat)/K-m)(app) of 1.8 x 10(2) (mole/titer)(-1) second(-1), thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.