Semi-rational engineering of a novel halohydrin dehalogenase from Sneathiella limimaris for the enantioselective synthesis of (S)-5-phenoxymethyl-2-oxazolidinone

Abstract

Enantiomerically pure 2-oxazolidinones are widely used as chiral auxiliaries in organic synthesis, but there is an unmet need for more effective methods to access these compounds. Here we report the identification and semi-rational engineering of the halohydrin dehalogenase SlHHDH from Sneathiella limimaris for the highly enantioselective ring-opening of phenyl glycidyl ether (PGE) with cyanate to yield (S)-5-phenoxymethyl-2-oxazolidinone. After single and combinatorial mutagenesis, the best enantioselective triple mutant, F15W/A137T/N179L achieved an enantioselectivity of 97 %, with an E value of 154. In addition, it could accept a wider range of PGEs to generate corresponding (S)-5-phenoxymethyl-2-oxazolidinones, whereby the product ee values increased from less than 5 % in wild-type SlHHDH to between 81 and 96 % in the triple mutant. Structural analysis of SlHHDH and mutant F15W/A137T/N179L in complex with the substrate PGE showed that changes of the substrate-binding pocket in the mutant position R-PGE farther from the catalytic residues, which may explain the enhanced enantioselectivity. This mutant has great potential as a biocatalyst for the first synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinones.