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Research article - Peer-reviewed, 2009

Directed Evolution of an Enantioselective Epoxide Hydrolase: Uncovering the Source of Enantioselectivity at Each Evolutionary Stage

Reetz, Manfred T.; Bocola, Marco; Wang, Li-Wen; Sanchis, Joaquin; Cronin, Annette; Arand, Michael; Zou, Jinyu; Archelas, Alain; Bottalla, Anne-Lise; Naworyta, Agata; Mowbray, Sherry L.


Directed evolution of enzymes as enantioselective catalysts in organic chemistry is an alternative to traditional asymmetric catalysis using chiral transition-metal complexes or organocatalysts, the different approaches often being complementary. Moreover, directed evolution studies allow us to learn more about how enzymes perform mechanistically. The present study concerns a previously evolved highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger in the hydrolytic kinetic resolution of racemic glycidyl phenyl ether. Kinetic data, molecular dynamics calculations, molecular modeling, inhibition experiments, and X-ray structural work for the wild-type (WT) enzyme and the best mutant reveal the basis of the large increase in enantioselectivity (E = 4.6 versus E = 115). The overall structures of the WT and the mutant are essentially identical, but dramatic differences are observed in the active site as revealed by the X-ray structures. All of the experimental and computational results support a model in which productive positioning of the preferred (S)-glycidyl phenyl ether, but not the (R)-enantiomer, forms the basis of enhanced enantioselectivity. Predictions regarding substrate scope and enantioselectivity of the best mutant are shown to be possible.

Published in

Journal of the American Chemical Society
2009, volume: 131, number: 21, pages: 7334-7343

Authors' information

Reetz, Manfred T.
Max Planck Institute for Coal Research
Bocola, Marco
Wang, Li-Wen
Max Planck Institute for Coal Research
Sanchis, Joaquin
Max Planck Institute for Coal Research
Cronin, Annette
University of Zürich
Arand, Michael
University of Zürich
Zou, Jinyu
Uppsala University
Archelas, Alain
University Paul Cezanne
Bottalla, Anne-Lise
Naworyta, Agata
Swedish University of Agricultural Sciences, Department of Molecular Biology
Mowbray, Sherry
Swedish University of Agricultural Sciences, Department of Molecular Biology

UKÄ Subject classification

Biochemistry and Molecular Biology

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