HheG, a Halohydrin Dehalogenase with Activity on Cyclic Epoxides

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 angstrom. 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.