Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue

Bert van Loo, Jaap Kingma, Gertjan Heyman, Alex Wittenaar, Jeffrey H. Lutje Spelberg, Theo Sonke, Dick B. Janssen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

32 Citations (Scopus)

Abstract

In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee.

Original languageEnglish
Pages (from-to)145-153
Number of pages9
JournalEnzyme and Microbial Technology
Volume44
Issue number3
Early online date17 Oct 2008
DOIs
Publication statusPublished - 5 Mar 2009
Externally publishedYes

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