Molecular Trick to Reverse SN2 Step in a Haloalkane Dehalogenase
Authors
Toul, M., Marques, S. M., Gao, T., Bernhardova, H., Vavra, O., Novakova, V., Damborsky, J., Bednar, D., Prokop, Z., Marek, M.
Source
CHEM CATALYSIS XXX: 101687 (2026)
Abstract
Hydrolytic haloalkane dehalogenase enzymes catalyze an SN2 nucleophilic substitution to erase halogen substituents in organohalogen compounds. The acid-base-nucleophile triad secures irreversible SN2 displacement of the halogen for the hydroxyl derived from the water. Catalysis relies on the protonatable imidazole ring of the histidine base, and its substitution with an asparagine traps the enzyme in a covalently bound intermediate state, a principle exploited in the widely used HaloTag technology. By contrast, the histidine-to-phenylalanine substitution triggers reversibility of the SN2 reaction, but the molecular trick by which it reprograms the catalytic pathway remains unknown. Here, we show that the phenylalanine at the site of the histidine base spatially disturbs the adjacent residues, leading to the remodeling of surrounding active-site loops. Consequently, rerouting the access tunnels imparts distinctive kinetic behavior featuring a reversible SN2 chemical step that facilitates transhalogenation reactions. This information is crucial for engineering next-generation biocatalysts for sustainable chemistry.