title: Second-Generation Engineering of a Thermostable Transketolase (TKGst) 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 TKGstbased 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 TKGstfor various applications in asymmetric carboligation.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, 200237, Shanghai, P.R. China.
authors.forename: Chaoqiang
authors.initials: C
authors.lastname: Zhou
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: Université Blaise Pascal ou Université Clermont Auvergne, Institut de Chimie de Clermont-Ferrand, B. P. 10448, 63000, Clermont-Ferrand, France.|CNRS, UMR 6296, ICCF, 63177, Aubière, France.
authors.forename: Marion
authors.initials: M
authors.lastname: Lorillière
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, 200237, Shanghai, P.R. China.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
authors.affiliation: Université Blaise Pascal ou Université Clermont Auvergne, Institut de Chimie de Clermont-Ferrand, B. P. 10448, 63000, Clermont-Ferrand, France.|CNRS, UMR 6296, ICCF, 63177, Aubière, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Charmantray
authors.affiliation: Université Blaise Pascal ou Université Clermont Auvergne, Institut de Chimie de Clermont-Ferrand, B. P. 10448, 63000, Clermont-Ferrand, France.|CNRS, UMR 6296, ICCF, 63177, Aubière, 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
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, 200237, Shanghai, P.R. China.
authors.forename: Dong
authors.identifier: 0000-0003-2594-0464
authors.initials: D
authors.lastname: Yi
mesh_terms: D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D004795:Enzyme Stability; D008954:Models, Biological; D015394:Molecular Structure; D015202:Protein Engineering; D013237:Stereoisomerism; D013696:Temperature; D014174:Transketolase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D014174:Transketolase
keywords: biocatalysis; carboligation; directed evolution; protein engineering; transketolases