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Position 123 of halohydrin dehalogenase HheG plays an important role in stability, activity, and enantioselectivity.

ABSTRACT: HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at position 123 were identified which displayed up to 14 °C increased apparent melting temperatures and up to three-fold higher activity. Aromatic amino acids at position 123 resulted even in a slightly higher enantioselectivity. Crystal structures of variants T123W and T123G revealed a flexible loop opposite to amino acid 123. In variant T123G, this loop adopted two different positions resulting in an open or partially closed active site. Classical molecular dynamics simulations confirmed a high mobility of this loop. Moreover, in variant T123G this loop adopted a position much closer to residue 123 resulting in denser packing and increased buried surface area. Our results indicate an important role for position 123 in HheG and give first structural and mechanistic insight into the thermostabilizing effect of mutations T123W and T123G.

SUBMITTER: Solarczek J

PROVIDER: S-EPMC6434027 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Position 123 of halohydrin dehalogenase HheG plays an important role in stability, activity, and enantioselectivity.

Solarczek Jennifer J Klünemann Thomas T Brandt Felix F Schrepfer Patrick P Wolter Mario M Jacob Christoph R CR Blankenfeldt Wulf W Schallmey Anett A

Scientific reports 20190325 1

HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at ...[more]

PMID: 30911023

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Project description:Halohydrin dehalogenase HheG and its homologues are remarkable enzymes for the efficient ring opening of sterically demanding internal epoxides using a variety of nucleophiles. The enantioselectivity of the respective wild-type enzymes, however, is usually insufficient for application and frequently requires improvement by protein engineering. We herein demonstrate that the highly flexible N-terminal loop of HheG, comprising residues 39 to 47, has a tremendous impact on the activity as well as enantioselectivity of this enzyme in the ring opening of structurally diverse epoxide substrates. Thus, highly active and enantioselective HheG variants could be accessed through targeted engineering of this loop. In this regard, variant M45F displayed almost 10-fold higher specific activity than wild type in the azidolysis of cyclohexene oxide, yielding the corresponding product (1S,2S)-2-azidocyclohexan-1-ol in 96%eeP (in comparison to 49%eeP for HheG wild type). Moreover, this variant was also improved regarding activity and enantioselectivity in the ring opening of cyclohexene oxide with other nucleophiles, demonstrating even inverted enantioselectivity with cyanide and cyanate. In contrast, a complete loop deletion yielded an inactive enzyme. Concomitant computational analyses of HheG M45F in comparison to wild type enzyme revealed that mutation M45F promotes the productive binding of cyclohexene oxide and azide in the active site by establishing noncovalent C-H ··π interactions between epoxide and F45. These interactions further position one of the two carbon atoms of the epoxide ring closer to the azide, resulting in higher enantioselectivity. Additionally, stable and enantioselective cross-linked enzyme crystals of HheG M45F were successfully generated after combination with mutation D114C. Overall, our study highlights that a highly flexible loop in HheG governs the enzyme's activity and selectivity in epoxide ring opening and should thus be considered in future protein engineering campaigns of HheG.

Dataset Information

Position 123 of halohydrin dehalogenase HheG plays an important role in stability, activity, and enantioselectivity.

Publications

Position 123 of halohydrin dehalogenase HheG plays an important role in stability, activity, and enantioselectivity.

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