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Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single‐Point Mutations
Author(s) -
Smith Mark E. B.,
Hibbert Edward G.,
Jones Alexander B.,
Dalby Paul A.,
Hailes Helen C.
Publication year - 2008
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.200800489
Subject(s) - chemistry , stereoselectivity , transketolase , escherichia coli , mutant , enantiomer , point mutation , enantiomeric excess , stereochemistry , reversion , combinatorial chemistry , enantioselective synthesis , biochemistry , catalysis , gene , enzyme , phenotype
Chiral auxiliary methodology and chiral assays have been developed to establish the enantiomeric purities of erythrulose and 1,3‐dihydroxypentan‐2‐one generated using wild‐type (WT) Escherichia coli transketolase (TK). L ‐Erythrulose was formed in 95% ee and (3 S )‐1,3‐dihydroxypentan‐2‐one in 58% ee . Since the latter compound was formed in moderate ee , TK libraries were screened to identify higher performing mutants. A colorimetric screen and chiral assay were successfully applied to a 96‐well format, and new active TK mutants were identified, which gave 1,3‐dihydroxypentan‐2‐one in high stereoselectivities. Remarkably, active‐site single‐point mutants were identified that were able to both enhance and reverse the stereoselectivity of TK.