Biochemical and biocatalytic characterization of 17 novel halohydrin dehalogenases.

Halohydrin dehalogenases are rare but catalytically remarkable enzymes since they are able to form novel C-C, C-O, C-N, or C-S bonds. Very recently, a motif-based sequence database mining approach resulted in the identification of 37 novel halohydrin dehalogenase enzymes, many of them exhibiting only low sequence similarity to previously known halohydrin dehalogenases. In an attempt to explore the biocatalytic potential of these newly identified enzymes, 17 representatives from all six phylogenetic subtypes were heterologously produced in Escherichia coli, purified and characterized to determine their substrate scopes in the dehalogenation and epoxide ring-opening reaction. Several enzymes with broad substrate spectra were identified exhibiting high activities towards a selection of typical substrates. Moreover, four halohydrin dehalogenases were found to be significantly more thermostable than the previously known HheC from Agrobacterium radiobacter AD1. Investigation of the enzymes' stereoselectivity in the dehalogenation of racemic 2-chloro-1-phenylethanol revealed that their stereopreference correlates with the phylogenetic placing of the enzymes in subtypes A through G. Furthermore, the biocatalytic potential of these novel halohydrin dehalogenases was investigated in the preparation of ethyl 4-cyano-3-hydroxybutyrate, a statin side-chain precursor. Though none of the active enzymes selectively formed the required (R)-enantiomer, several halohydrin dehalogenases were identified with significantly higher activity in the conversion compared to HheC, making them promising candidates for this industrially relevant reaction.