Molecular trick to reverse S N 2 mechanism in hydrolytic enzyme
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Hydrolytic haloalkane dehalogenase enzymes catalyze an S N 2 nucleophilic substitution to erase halogen substituents in organohalogen compounds. The acid-base-nucleophile triad secures irreversible S N 2 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. In contrast, the histidine-to-phenylalanine substitution triggers reversibility of the S N 2 mechanism, 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 of the access tunnels imparts distinctive kinetic behavior, featuring a reversible S N 2 chemical step that facilitates transhalogenation reactions. This information is crucial for engineering next-generation biocatalysts for sustainable chemistry.
Highlights
-
Catalytic triad (glutamate-histidine-aspartate) secures irreversible S N 2 mechanism
-
Histidine-to-phenylalanine substitution in the triad leads to a reversible S N 2 reaction
-
Enzyme active site rerouting is a hallmark of the catalytic reprogramming
-
Basis for designing biocatalysts for sustainable transhalogenation chemistry
The bigger picture
Haloalkane dehalogenases catalyze the hydrolytic cleavage of the carbon-halogen bond in halogenated hydrocarbons, a feature used in a wide variety of industrial and biotechnological processes. In HaloTag technology, the catalytic histidine is replaced by an asparagine to enable a stable covalent bonding between a probe and a protein for biological imaging, affinity purification, etc. Remarkably, this originally irreversible S N 2 process becomes fully reversible in a histidine-to-phenylalanine mutant. Moreover, halogen ion product is released at this stage, offering a greener alternative for transhalogenation reactions compared to the conventional synthetic approach and a way to recycle environmentally harmful halogenated compounds. However, the optimization of enzymes is needed for applications on an industrial scale. Therefore, we investigated the kinetics of the S N 2 step and structural features of these two enzyme mutants, providing a basis for subsequent optimization efforts.
