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Reference: Ranoux A, et al. (2012) Enhancement of the substrate scope of transketolase. Chembiochem 13(13):1921-31

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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 28.8x10(-3) min(-1) for the wild-type) and 2.6 times more active for substrates LiHPA/rib.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Ranoux A, Karmee SK, Jin J, Bhaduri A, Caiazzo A, Arends IW, Hanefeld U
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