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- Title
Tailoring the S-Selectivity of 2-Succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate Synthase (MenD) from Escherichia coli.
- Authors
Westphal, Robert; Hahn, Doris; Mackfeld, Ursula; Waltzer, Simon; Beigi, Maryam; Widmann, Michael; Vogel, Constantin; Pleiss, Jürgen; Müller, Michael; Rother, Dörte; Pohl, Martina
- Abstract
The thiamine diphosphate (ThDP)-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase from Escherichia coli ( EcMenD, E.C. 2.2.1.9) catalyzes the carboligation of α-ketoglutarate (α-KG) and various benzaldehyde derivatives with excellent chemo- as well as high R-selectivity (enantiomeric excess ( ee) >93 %) to yield chiral α-hydroxy ketones. Based on the recently developed S-pocket concept, we engineered S-selective EcMenD variants by optimizing the steric properties and stabilization of the acceptor substrate in the S-pocket. Moreover, the moderate S-selectivity of the EcMenD variant I474A/F475G described recently for the carboligation of α-KG and benzaldehyde ( ee=75 %) could be improved by selective destabilization of the R-pathway, which resulted in the variant I474A/F475G/R395Y ( ee=85 % S). Subsequent investigation of the acceptor substrate range of this new variant revealed high S-selectivity especially with meta-substituted benzaldehydes, which gave access to 5-hydroxy-4-oxo-5-arylpentanoates with excellent enantioselectivities of up to 99 % ee S. Thus, opening the S-pocket and simultaneous destabilization of the R-pathway provides a potential general new strategy to enhance the S-selectivity of ThDP-dependent enzymes.
- Subjects
THIAMIN pyrophosphate; ESCHERICHIA coli enzymes; BENZALDEHYDE; PROTEIN engineering; COUPLING reactions (Chemistry)
- Publication
ChemCatChem, 2013, Vol 5, Issue 12, p3587
- ISSN
1867-3880
- Publication type
Article
- DOI
10.1002/cctc.201300318