Altering the Substrate Specificity of Reductase CgKR1 from Candida glabrata by Protein Engineering for Bioreduction of Aromatic α-Keto Esters

A versatile keto ester reductase CgKR1, exhibiting a broad substrate spectrum, was obtained from Candida glabrata by genome data mining. It showed the highest activity toward an aliphatic β‐keto ester, ethyl 4‐chloro‐3‐oxobutanoate (COBE), but much lower activity toward bulkier α‐keto esters with an...

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Bibliographic Details
Published in:Advanced synthesis & catalysis 2014-06, Vol.356 (9), p.1943-1948
Main Authors: Huang, Lei, Ma, Hong-Min, Yu, Hui-Lei, Xu, Jian-He
Format: Article
Language:English
Subjects:
aromatic α-keto esters
protein engineering
reductases
substrate specificity
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Summary:A versatile keto ester reductase CgKR1, exhibiting a broad substrate spectrum, was obtained from Candida glabrata by genome data mining. It showed the highest activity toward an aliphatic β‐keto ester, ethyl 4‐chloro‐3‐oxobutanoate (COBE), but much lower activity toward bulkier α‐keto esters with an aromatic group, such as methyl ortho‐chlorobenzoylformate (CBFM) and ethyl 2‐oxo‐4‐phenylbutyrate (OPBE). By rational design of the active pocket, the substrate specificity of the reductase was significantly altered and this tailor‐made reductase showed a much higher activity toward aromatic α‐keto esters (∼7‐fold increase in kcat/Km toward CBFM) and lower activity toward aliphatic keto esters (∼12‐fold decrease in kcat/Km toward COBE). Meanwhile, the thermostability of the reductase was enhanced by a consensus approach. Such improvements may yield practical catalysts for the asymmetric bioreduction of these aromatic α‐keto esters
ISSN:1615-4150
1615-4169
DOI:10.1002/adsc.201300775