Insights into the molecular determinants of thermal stability in halohydrin dehalogenase HheD2

Halohydrin dehalogenases (HHDHs) are promising enzymes for application in biocatalysis due to their promiscuous epoxide ring‐opening activity with various anionic nucleophiles. So far, seven different HHDH subtypes A to G have been reported with subtype D containing the by far largest number of enzymes. Moreover, several characterized members of subtype D have been reported to display outstanding characteristics such as high catalytic activity, broad substrate spectra or remarkable thermal stability. Yet, no structure of a D‐type HHDH has been reported to date that could be used to investigate and understand those features on a molecular level. We therefore solved the crystal structure of HheD2 from gamma proteobacterium HTCC2207 at 1.6 Å resolution and used it as a starting point for targeted mutagenesis in combination with molecular dynamics (MD) simulation, in order to study the low thermal stability of HheD2 in comparison with other members of subtype D. This revealed a hydrogen bond between conserved residues Q160 and D198 to be connected with a high catalytic activity of this enzyme. Moreover, a flexible surface region containing two α‐helices was identified to impact thermal stability of HheD2. Exchange of this surface region by residues of HheD3 yielded a variant with 10 °C higher melting temperature and reaction temperature optimum. Overall, our results provide important insights into the structure–function relationship of HheD2 and presumably for other D‐type HHDHs. Databases Structural data are available in PDB database under the accession number 7B73. Thermostabilization of halohydrin dehalogenase (HHDH) HheD2 by protein engineering in combination with molecular dynamics simulations revealed important insights into the structure–function relationship of D‐type HHDHs. While wild‐type HheD2 inactivates at temperatures above 45 °C, exchange of a flexible surface region in HheD2 by residues of the more thermostable HHDH HheD3 resulted in a HheD2 variant retaining high catalytic activity at 60 °C.