Characterization of a new G-type halohydrin dehalogenase with enhanced catalytic activity

Günther, Sophie; Schallmey, Anett

doi:10.26434/chemrxiv-2022-p9rzx

2022

DOI: 10.26434/chemrxiv-2022-p9rzx

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Characterization of a new G-type halohydrin dehalogenase with enhanced catalytic activity

Sophie Günther

Anett Schallmey

Abstract: Biochemical characterization of HheG-682, a new homolog of halohydrin dehalogenase HheG sharing 46% sequence identity, is presented.

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2024

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A Dynamic Loop in Halohydrin Dehalogenase HheG Regulates Activity and Enantioselectivity in Epoxide Ring Opening

Staar,

Ahlborn,

Estévez-Gay

et al. 2024

ACS Catal.

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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.

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A Dynamic Loop in Halohydrin Dehalogenase HheG Regulates Activity and Enantioselectivity in Epoxide Ring Opening

Staar,

Ahlborn,

Estévez-Gay

et al. 2024

ACS Catal.

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Characterization of a new G-type halohydrin dehalogenase with enhanced catalytic activity

Abstract: Biochemical characterization of HheG-682, a new homolog of halohydrin dehalogenase HheG sharing 46% sequence identity, is presented.

Cited by 1 publication

References 12 publications

A Dynamic Loop in Halohydrin Dehalogenase HheG Regulates Activity and Enantioselectivity in Epoxide Ring Opening

A Dynamic Loop in Halohydrin Dehalogenase HheG Regulates Activity and Enantioselectivity in Epoxide Ring Opening

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