Second-Generation Engineering of a Thermostable Transketolase (TK Gst ) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity

Abstract:

The transketolase from Geobacillus stearothermophilus (TKGst) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non‐α‐hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TKGst based on double‐site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TKGst for various applications in asymmetric carboligation.

SEEK ID: https://fairdomhub.org/publications/416

DOI: 10.1002/cbic.201600609

Projects: TRALAMINOL

Journal: ChemBioChem

Citation: ChemBioChem 18(5) : 455

Date Published: 2nd Mar 2017

Authors: Chaoqiang Zhou, Thangavelu Saravanan, Marion Lorillière, Dongzhi Wei, Franck Charmantray, Laurence Hecquet, Wolf Dieter Fessner, Dong Yi

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Zhou, C., Saravanan, T., Lorillière, M., Wei, D., Charmantray, F., Hecquet, L., … Yi, D. (2017). Second-Generation Engineering of a Thermostable Transketolase (TKGst) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity. ChemBioChem, 18(5), 455–459. http://doi.org/10.1002/cbic.201600609
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Created: 28th Jun 2019 at 11:29

Last updated: 28th Jun 2019 at 11:30

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