Semi‐rational protein engineering of a novel ene‐reductase from Galdieria sulphuraria for asymmetric reduction of (R)‐carvone and ketoisophorone

Asymmetric reduction of (R)‐carvone and ketoisophorone by an engineered ene‐reductase from Galdieria sulphuraria (GsOYE) combined with glucose dehydrogenase for NADPH regeneration were studied. A semi‐rational protein engineering was used to enhance the activity and selectivity of GsOYE. Upon the se...

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Bibliographic Details
Published in:Biotechnology and applied biochemistry 2023-04, Vol.70 (2), p.697-706
Main Authors: Wu, Shijin, Wang, Bijiao, Yan, Hongde
Format: Article
Language:English
Subjects:
alga
Amino acid sequence
Amino acids
Asymmetry
Biocatalysis
Carvone
ene‐reductase
Galdieria sulphuraria
Glucose dehydrogenase
Molecular docking
Molecular Docking Simulation
NADPH dehydrogenase
Nucleotide sequence
old yellow enzyme
Oxidoreductases - metabolism
Protein Engineering
Proteins
Reductases
Reduction
Stereoselectivity
Substitutes
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Summary:Asymmetric reduction of (R)‐carvone and ketoisophorone by an engineered ene‐reductase from Galdieria sulphuraria (GsOYE) combined with glucose dehydrogenase for NADPH regeneration were studied. A semi‐rational protein engineering was used to enhance the activity and selectivity of GsOYE. Upon the sequence alignment and molecular docking results, two amino acid residues at positions 66 and 270 were selected as saturation mutation sites. Finally, a single substitution variant of GsOYE‐N270A with complete conversion (100%) and diastereoselectivity (dep>99%) for reduction of (R)‐carvone and a double substitution variant GsOYE‐Y66P/N270H with improved stereoselectivity for reduction of ketoisophorone were obtained. Graphical
ISSN:0885-4513
1470-8744
DOI:10.1002/bab.2391