2026-04-14T07:19:04Zhttps://recercat.cat/oai/request
oai:recercat.cat:10256/191882024-06-18T13:56:07Zcom_2072_452955com_2072_2054col_2072_453073
RECERCAT author Wessel, Julia author Petrillo, Giovanna author Estévez Gay, Miquel author Bosch, Sandra author Seeger, Margarita author Dijkman, Willem P. author Iglesias-Fernández, Javier author Hidalgo, Aurelio author Uson, Isabel author Osuna Oliveras, Sílvia author Schallmey, Anett 2024-06-18T13:56:07Z 2024-06-18T13:56:07Z 2021-02-19 http://hdl.handle.net/10256/19188 Halohydrin dehalogenases (HHDHs) are promising enzymes for application in biocatalysis due to their promiscuous epoxide ring opening activity with various anionic nucleophiles. So far, 7 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 halohydrin dehalogenase 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 to 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 as well as reaction temperature optimum. Overall, our results provide important insights into the structure‐function relationship of HheD2 and presumably for other D‐type halohydrin dehalogenasesThis work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 635595 (CARBAZYMES). This work was further supported by grants PGC2018-101370-B-100 (the Spanish Ministry of Science and Innovation and EU FEDER fonds) and the Generalitat de Catalunya (2017SGR-1192). We also thank the Generalitat de Catalunya for the emerging group CompBioLab (2017 SGR-1707) and Spanish MINECO for project PGC2018-102192-BI00. J.I.F. was supported by the European Community for Marie Curie fellowship (H2020-MSCA-IF-2016- 753045) and Juan de la Cierva-Incorporación fellowship (IJCI-2017-34129). S.O. is grateful to the funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-2015-StG-679001) Reconeixement 4.0 Internacional http://creativecommons.org/licenses/by/4.0 info:eu-repo/semantics/openAccess Enzims Enzymes Dinàmica molecular Molecular dynamics Insights into the molecular determinants of thermal stability in halohydrin dehalogenase HheD2 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion peer-reviewed