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Donor Promiscuity of a Thermostable Transketolase by Directed Evolution: Efficient Complementation of 1‐Deoxy‐ d ‐xylulose‐5‐phosphate Synthase Activity
Author(s) -
Saravanan Thangavelu,
Junker Sebastian,
Kickstein Michael,
Hein Sascha,
Link MarieKristin,
Ranglack Jan,
Witt Samantha,
Lorillière Marion,
Hecquet Laurence,
Fessner WolfDieter
Publication year - 2017
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201701169
Subject(s) - transketolase , complementation , directed evolution , ketose , biochemistry , chemistry , enzyme , auxotrophy , lyase , mutant , escherichia coli , biosynthesis , stereochemistry , gene , aldose , glycoside
Enzymes catalyzing asymmetric carboligation reactions typically show very high substrate specificity for their nucleophilic donor substrate components. Structure‐guided engineering of the thermostable transketolase from Geobacillus stearothermophilus by directed in vitro evolution yielded new enzyme variants that are able to utilize pyruvate and higher aliphatic homologues as nucleophilic components for acyl transfer instead of the natural polyhydroxylated ketose phosphates or hydroxypyruvate. The single mutant H102T proved the best hit toward 3‐methyl‐2‐oxobutyrate as donor, while the double variant H102L/H474S showed highest catalytic efficiency toward pyruvate as donor. The latter variant was able to complement the auxotrophic deficiency of Escherichia coli cells arising from a deletion of the dxs gene, which encodes for activity of the first committed step into the terpenoid biosynthesis, offering the chance to employ a growth selection test for further enzyme optimization.