Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis |
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Authors: | Khare Sagar D Kipnis Yakov Greisen Per Takeuchi Ryo Ashani Yacov Goldsmith Moshe Song Yifan Gallaher Jasmine L Silman Israel Leader Haim Sussman Joel L Stoddard Barry L Tawfik Dan S Baker David |
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Affiliation: | Department of Biochemistry, University of Washington, Seattle, Washington, USA. |
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Abstract: | 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. |
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