Halohydrin dehalogenases (HHDHs) are of biotechnological interest due to their promiscuous epoxide ring-opening activity with a set of negatively charged nucleophiles, enabling the formation of C−C, C−N, or C−O bonds. The recent discovery of HHDH-specific sequence motifs aided the identification of a large number of halohydrin dehalogenases from public sequence databases, enlarging the biocatalytic toolbox substantially. During the characterization of 17 representatives of these phylogenetically diverse enzymes, one HHDH, namely HheG from Ilumatobacter coccineus, was identified to convert cyclic epoxide substrates. The enzyme exhibits significant activity in the azidolysis of cyclohexene oxide and limonene oxide with turnover numbers of 7.8 and 44 s −1 , respectively. As observed for other HHDHs, the cyanide-mediated epoxide ring-opening proceeded with lower rates. Wild-type HheG displays modest enantioselectivity, as the resulting azido-and cyanoalcohols of cyclohexene oxide ring-opening were obtained in 40% enantiomeric excess. These biocatalytic findings were further complemented by the crystal structure of the enzyme refined to 2.3 Å. Analysis of HheG's structure revealed a large open cleft harboring the active site. This is in sharp contrast to other known HHDH structures and aids in explaining the special substrate scope of HheG.
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.
bHalohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community.
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