Beier_2019_Adv.Synth.Catal_361_2438

Haloalkane dehalogenases (HLDs) are a/beta-hydrolases that convert halogenated compounds to their corresponding alcohols. The overall kinetic mechanism proceeds via four steps: (i) binding of halogenated substrate, (ii) bimolecular nucleophilic substitution (SN2) leading to the cleavage of a carbon-halogen bond and the formation of an alkyl-enzyme intermediate, (iii) nucleophilic addition of a water molecule resulting in the hydrolysis of the intermediate to the corresponding alcohol and (iv) release of the reaction products an alcohol, a halide ion and a proton. Although, the overall reaction has been reported as irreversible, several kinetic evidences from previous studies suggest the reversibility of the first SN2 chemical step. To study this phenomenon, we have engineered HLDs to stop the catalytic cycle at the stage of the alkyl-enzyme intermediate. The ability of the intermediate to exchange halides was confirmed by a stopped-flow fluorescence binding analysis. Finally, the transhalogenation reaction was confirmed with several HLDs and 2,3-dichloropropene in the presence of a high concentration of iodide. The formation of the transhalogenation product 3-iodo-2-chloropropene catalysed by five mutant HLDs was identified by gas chromatography coupled with mass spectrometry. Hereby we demonstrated the reversibility of the cleavage of the carbon-halogen bond by HLDs resulting in a transhalogenation. After optimization, the transhalogenation reaction can possibly find its use in biocatalytic applications. Enabling this reaction by strategically engineering the enzyme to stop at an intermediate in the catalytic cycle that is synthetically more useful than the product of the natural pathway is a novel concept.