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Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code
Zhoutong Sun1,2, Richard Lonsdale1,2, Guangyue Li1,2
1Department of Synthetic Organic Chemistry, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim an der Ruhr, Germany.
Chembiochem : a European Journal of Chemical Biology
|July 14, 2016
Summary
Double-code saturation mutagenesis (DCSM) enhances enzyme engineering by using two amino acids to improve stereoselectivity, outperforming single-code methods. This protein engineering approach yields selective catalysts for valuable chemical transformations.
Area of Science:
- Enzyme engineering
- Protein engineering
- Biocatalysis
Background:
- Saturation mutagenesis is a protein engineering technique for altering enzyme stereoselectivity.
- Reduced amino acid alphabets and site splitting are used to manage screening complexity in mutagenesis.
- Limonene epoxide hydrolase (LEH) has been a model system for comparing single-code (SCSM) and triple-code (TCSM) saturation mutagenesis.
Purpose of the Study:
- To investigate the efficacy of double-code saturation mutagenesis (DCSM) as a protein engineering strategy.
- To compare DCSM with SCSM using cyclohexene oxide hydrolytic desymmetrization as a model reaction.
- To identify the structural basis for enantioselectivity using docking computations.
Main Methods:
- Employed double-code saturation mutagenesis (DCSM) with a two-amino-acid alphabet selected via rational design.
- Utilized hydrolytic desymmetrization of cyclohexene oxide to produce (R,R)- or (S,S)-cyclohexane-1,2-diol.
- Performed comparative analysis against single-code saturation mutagenesis (SCSM) and docking computations.
Main Results:
- DCSM demonstrated clear superiority over SCSM in generating stereoselective mutants.
- Achieved mutants capable of producing both (R,R)- and (S,S)-cyclohexane-1,2-diol.
- Developed variants exhibited catalytic activity with additional substrates, and docking revealed enantioselectivity determinants.
Conclusions:
- DCSM is an effective protein engineering method for enhancing enzyme stereoselectivity.
- This approach offers a powerful alternative to SCSM and TCSM for creating selective biocatalysts.
- Rational design of reduced amino acid alphabets is key to successful DCSM applications.
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