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Enhancement of the Substrate Scope of Transketolase
Author(s) -
Ranoux Adeline,
Karmee Sanjib K.,
Jin Jianfeng,
Bhaduri Anirban,
Caiazzo Aldo,
Arends Isabel W. C. E.,
Hanefeld Ulf
Publication year - 2012
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201200240
Subject(s) - transketolase , glycolaldehyde , chemistry , directed evolution , mutant , mutagenesis , enzyme , active site , biochemistry , biocatalysis , combinatorial chemistry , in silico , docking (animal) , stereochemistry , catalysis , ionic liquid , gene , medicine , nursing
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.