title: Donor Promiscuity of a Thermostable Transketolase by Directed Evolution: Efficient Complementation of 1-Deoxy-d-xylulose-5-phosphate Synthase Activity.
abstract: 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.
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
authors.forename: Thangavelu
authors.initials: T
authors.lastname: Saravanan
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Junker
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Kickstein
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Hein
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
authors.forename: Marie-Kristin
authors.initials: MK
authors.lastname: Link
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Ranglack
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
authors.forename: Samantha
authors.initials: S
authors.lastname: Witt
authors.affiliation: Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut de Chimie de Clermont-Ferrand (ICCF), F-, 63000, Clermont-Ferrand, France.
authors.forename: Marion
authors.initials: M
authors.lastname: Lorillière
authors.affiliation: Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut de Chimie de Clermont-Ferrand (ICCF), F-, 63000, Clermont-Ferrand, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Hecquet
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
authors.forename: Wolf-Dieter
authors.initials: WD
authors.lastname: Fessner
mesh_terms: D055162:Biocatalysis; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007651:Keto Acids / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D013696:Temperature; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014174:Transketolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D007651:Keto Acids; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D014174:Transketolase
keywords: acyloins; asymmetric synthesis; biocatalysis; carboligation; protein engineering