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  Reshaping an Enzyme Binding Pocket for Enhanced and Inverted Stereoselectivity: Use of Smallest Amino Acid Alphabets in Directed Evolution

Sun, Z., Lonsdale, R., Kong, X.-D., Xu, J.-H., Zhou, J., & Reetz, M. T. (2015). Reshaping an Enzyme Binding Pocket for Enhanced and Inverted Stereoselectivity: Use of Smallest Amino Acid Alphabets in Directed Evolution. Angewandte Chemie International Edition, 54(42), 12410-12415. doi:10.1002/anie.201501809.

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 Creators:
Sun, Zhoutong1, 2, Author              
Lonsdale, Richard1, 2, Author              
Kong, Xu-Dong3, Author
Xu, Jian-He3, Author
Zhou, Jiahai4, Author
Reetz, Manfred T.1, 2, Author              
Affiliations:
1Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
2Philipps-Universität Marburg, Fachbereich Chemie, ou_persistent22              
3East China University of Science and Technology, State Key Laboratory of Bioreactor Engineering, Shanghai 200237 , ou_persistent22              
4Chinese Academy of Sciences, Shanghai Institute of Organic Chemistry, State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai 200032 , ou_persistent22              

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Free keywords: amino acid alphabet; directed evolution; epoxide hydrolases; saturation mutagenesis; stereoselectivity
 Abstract: Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.

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Language(s): eng - English
 Dates: 2015-02-272015-03-182015-04-172015-10-12
 Publication Status: Published in print
 Pages: 6
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1002/anie.201501809
 Degree: -

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Title: Angewandte Chemie International Edition
  Abbreviation : Angew. Chem. Int. Ed.
Source Genre: Journal
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Pages: 6 Volume / Issue: 54 (42) Sequence Number: - Start / End Page: 12410 - 12415 Identifier: ISSN: 1521-3773
CoNE: https://pure.mpg.de/cone/journals/resource/0570-0833