Jiang Lin, Althoff Eric A, Clemente Fernando R, Doyle Lindsey, Röthlisberger Daniela, Zanghellini Alexandre, Gallaher Jasmine L, Betker Jamie L, Tanaka Fujie, Barbas Carlos F, Hilvert Donald, Houk Kendall N, Stoddard Barry L, Baker David
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Science. 2008 Mar 7;319(5868):1387-91. doi: 10.1126/science.1152692.
The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.
设计出能够催化任何所需化学反应的酶，是计算蛋白质设计面临的一项重大挑战。我们利用依赖哈希技术构建多步反应活性位点的新算法，设计了逆向醛缩酶，这些酶使用四种不同的催化基序来催化非天然底物中碳-碳键的断裂。在经实验表征的72种设计中，有32种（涵盖一系列蛋白质折叠类型）具有可检测到的逆向醛缩酶活性。与涉及带电侧链网络的设计相比，使用明确水分子介导质子转移的设计要成功得多，其速率加速高达四个数量级且能多次周转。嵌入两种蛋白质支架中的活性设计的X射线晶体结构证实了设计过程的原子精度，这两种晶体结构几乎都与设计模型重叠。
