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

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• 作者：Manfred T. Reetz ; Marco Bocola ; Li-Wen Wang ; Joaquin Sanchis ; Annette Cronin ; Michael Arand ; Jinyu Zou ; Alain Archelas ; Anne-Lise Bottalla ; Agata Naworyta ; Sherry L. Mowbray
• 刊名：Journal of the American Chemical Society
• 出版年：2009
• 出版时间：June 3, 2009
• 年：2009
• 卷：131
• 期：21
• 页码：7334-7343
• 全文大小：581K
• 年卷期：v.131,no.21(June 3, 2009)
• ISSN：1520-5126

文摘

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.