Department of Biochemistry, University of Washington, Seattle, Washington, USA.
Nat Chem Biol. 2012 Feb 5;8(3):294-300. doi: 10.1038/nchembio.777.
The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.
重新设计酶以催化非同源化学转化的能力将具有广泛的应用。我们开发了一种计算方法，用于重新利用金属酶活性位点官能团的反应性来催化新反应。使用这种方法，我们设计了一种含有锌的小鼠腺苷脱氨酶，使其能够在定向进化后以 ~10(4) M(-1) s(-1) 的催化效率（k(cat)/K(m)）催化模型有机膦的水解。在酶的高分辨率晶体结构中，除一个残基外，所有设计的残基都采用了设计的构象。设计的酶有效地催化神经毒剂沙林类似物香豆素基 R(P)异构体的水解，并且对香豆素离去基团表现出明显的底物选择性。天然酶活性位点的计算重新设计补充了定向进化方法，并为探索其用于新反应的未开发催化潜力提供了一种通用方法。
