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  Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis

Sun, Z., Lonsdale, R., Li, G., & Reetz, M. T. (2016). Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis. Chembiochem, 17(19), 1865-1872. doi:10.1002/cbic.201600296.

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 Creators:
Sun, Zhoutong1, Author              
Lonsdale, Richard1, Author              
Li, Guangyue1, Author              
Reetz, Manfred T.2, Author              
Affiliations:
1Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
2Philipps-Universität Marburg, Fachbereich Chemie, ou_persistent22              

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Free keywords: directed evolution · epoxide hydrolases · reduced amino acid alphabet · saturation mutagenesis · stereoselectivity
 Abstract: Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversamplin g in the screening step (the bottleneck) increases astronomically as the number of residues in the ran- domization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental plat- form in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenes is (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double-code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)- or (S,S)-cyclohexane- 1,2-diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R-andS,S-selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantio- selectivity.

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Language(s): eng - English
 Dates: 2016-08-162016-10-04
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1002/cbic.201600296
 Degree: -

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Title: Chembiochem
  Abbreviation : Chembiochem
Source Genre: Journal
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Pages: - Volume / Issue: 17 (19) Sequence Number: - Start / End Page: 1865 - 1872 Identifier: ISSN: 1439-4227
CoNE: https://pure.mpg.de/cone/journals/resource/110978984568897