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Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis


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 (kcat/Km) of 104 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 RP 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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Figure 1: Computational active site redesign.
Figure 2: Kinetic characterization of PT3 with DECP.
Figure 3: Spatial clustering of wild-type and activity-enhancing residues.
Figure 4: Design model and crystal structure of apoPT3.1.
Figure 5: PT3 variants stereo-selectively catalyze the hydrolysis of a cyclosarin analog.

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We thank L. Nivon for assistance with liquid chromatography, O. Khersonsky (Weizmann Institute of Science) for providing substrates and J. Damborsky for comments on the manuscript. This work was supported by the Defense Advanced Research Projects Agency, the Defense Threat Reduction Agency and the Howard Hughes Medical Institute. P.J.G. was supported by Novo Nordisk Danmark-Amerika Fondet and Oticon Fonden.

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Authors and Affiliations



S.D.K. developed the computational method for active site redesign, performed computational design and kinetic characterization of PT1–PT12, analyzed the data and wrote the paper. Y.K. designed and performed the directed evolution and library screening, wild-type activity measurements, substrate selectivity and inhibition experiments and analyzed the data and wrote the paper. P.J.G. implemented the computational redesign method, performed the computational design of PT1–PT12 and analyzed the data. R.T. determined the crystal structure of PT3.1. J.L.G. expressed and purified the designed proteins PT1–PT12. Y.S. performed pKa calculations. Y.A., M.G., I.S., H.L. and J.L.S. synthesized nerve agents and nerve agent analogues, screened these with PT3 and determined their stereoselectivity. B.L.S. performed structural analysis and wrote the paper. D.S.T. designed the experiments and analyzed the data. D.B. designed the computational method and the experiments, analyzed the data and wrote the paper.

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Correspondence to David Baker.

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Khare, S., Kipnis, Y., Greisen, P. et al. Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis. Nat Chem Biol 8, 294–300 (2012).

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