title: Enhancement of the substrate scope of transketolase.
abstract: To enhance the activity of transketolase towards nonphosphorylated substrates and enlarge the scope of its substrates, notably to long polyol aldehyde acceptors (D-ribose or D-glucose), a rational design-supported evolution strategy was applied. By using docking experiments, an in silico library, and iterative mutagenesis, libraries of single- and double-point mutants were designed and generated. A double-screening approach was implemented, coupling a preselection activity assay (HPLC method) and a selective assay (GC method) to find the best enzymes. Several mutants (R526N, R526Q, R526Q/S525T, R526K/S525T) showed improved activities towards nonphosphorylated substrates as the coupled products of lithium hydroxypyruvate (HPA) with glycolaldehyde (GO), D-ribose or D-glucose. These mutated enzymes were further characterised. They were shown to be up to four times more active than the wild-type (mutant R526Q/S525T) for nonphosphorylated substrates LiHPA/GO (V(m) /K(m)  for LiHPA = 92.4 instead of 28.8×10(-3)  min(-1)  for the wild-type) and 2.6 times more active for substrates LiHPA/rib.
authors: Technische Universiteit Delft, Gebouw voor Scheikunde, Afdeling Biotechnologie, Julianalaan 136, Delft, The Netherlands.
authors: Adeline
authors: A
authors: Ranoux
authors: Sanjib K
authors: SK
authors: Karmee
authors: Jianfeng
authors: J
authors: Jin
authors: Anirban
authors: A
authors: Bhaduri
authors: Aldo
authors: A
authors: Caiazzo
authors: Isabel W C E
authors: IW
authors: Arends
authors: Ulf
authors: U
authors: Hanefeld
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D019295:Computational Biology; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D011773:Pyruvates / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012266:Ribose / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014174:Transketolase / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011773:Pyruvates; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012266:Ribose; C012375:hydroxypyruvic acid; D014174:Transketolase; D000079:Acetaldehyde; D005947:Glucose; C010972:glycolaldehyde