Biochemical and biocatalytic characterization of 17 novel halohydrin dehalogenases

Abstract

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.

Selective Ring‐Opening of Di‐Substituted Epoxides Catalysed by Halohydrin Dehalogenases

“…HheG from Ilumatobacter coccineus was active on all five epoxide substrates yielding highest conversion for substrates 1 , 3 , 4 and 5 among all tested HHDHs. Significant conversion of 5 by HheG was unexpected as the enzyme was shown to exhibit only very little activity on styrene oxide . For HheA2 and HheB2, hardly any product formation in the conversion of the tested vicinally di‐substituted epoxides was observed, which is in agreement with previous findings …”

“…This is also supported by calculations of the active site volume of HheG, which was found to be 2.7 times larger than the calculated active site volume of HheC . On the other hand, epoxide substrates with much bulkier substituents on both carbon atoms of the epoxide ring are likely not accepted by HheG as it was previously shown that trans ‐stilbene oxide was not converted by HheG or other tested HHDHs …”

“…All HHDH enzymes except HheE4 and HheG2 were produced and purified as reported previously ,. Enzymes HheE4 and HheG2 were produced in E. coli BL21(DE3).…”

“…Expression cultures were grown at 22 °C for 24 h. Cells were harvested by centrifugation (4,400 g, 20 min at 4 °C) and cell pellets were stored at −20 °C until further use. Both enzymes were purified via affinity chromatography according to a previously published protocol . A list of all HHDHs used in this work with their respective source organisms and accession numbers is given in Table S2 in the supporting information.…”

“…We recently reported the identification of a large set of novel HHDH enzymes based on a database mining approach using HHDH‐specific sequence motifs . Together with the five previously known HHDHs, they compose a broad family of HHDH enzymes subdivided into six phylogenetic subtypes A through G. A representative subset of 17 novel HHDHs, which span the entire phylogenetic tree, was subsequently cloned and biocatalytically characterised . This way, HheG from Ilumatobacter coccineus was found to convert cyclic epoxides such as cyclohexene oxide and limonene oxide with synthetic useful activity .…”