3 years ago

Donor Promiscuity of a Thermostable Transketolase by Directed Evolution: Efficient Complementation of 1-Deoxy-d-xylulose-5-phosphate Synthase Activity

Donor Promiscuity of a Thermostable Transketolase by Directed Evolution: Efficient Complementation of 1-Deoxy-d-xylulose-5-phosphate Synthase Activity
Laurence Hecquet, Wolf-Dieter Fessner, Marie-Kristin Link, Michael Kickstein, Jan Ranglack, Thangavelu Saravanan, Sebastian Junker, Samantha Witt, Sascha Hein, Marion Lorillière
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. Structure-guided engineering of a bacterial transketolase yielded enzyme variants capable of using aliphatic oxoacids as non-natural nucleophilic components for stereospecific acyloin synthesis. The double variant H102L/H474S showed high catalytic efficiency toward pyruvate and was able to complement a deletion of the dxs gene coding for 1-deoxyxylulose-5-phosphate synthase, which catalyzes the first step of the terpenoid biosynthesis.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/anie.201701169

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