The Fate of Various Halogenated Organics in the Active Sites of LinB and DhaA Dehalogenases – A Combined Experimental and Computational Study

LinB and DhaA are well-known haloalkane dehalogenases (HLDs) capable of converting a plethora of halogenated alkanes, also those considered persistent pollutants. One way of studying the nature and efficiency of these important enzymes is to measure the kinetic isotope effect (KIE) on the metabolized reaction, in particular leaving group (either chloride or bromide) KIE. Although the general mechanism via which these two dehalogenating enzymes operate has been already studied and described, the isotope effects data providing information on the mechanistic details, especially Br KIE are scarce. In this work we aimed at gaining insights into the enzymatic dehalogenation of dibromo- and bromo-chloro- ethanes by LinB and DhaA, by combining experimental and computational methods. A model developed this way has been subsequently extended to dihalopropanes. Using the predicted free energy surface of the reaction catalyzed by HLD and kinetic isotope effects based on QM/MM calculations it has been demonstrated that with respect to the magnitudes of Br KIE conversion of 1,2-dibromoethane, 1-bromo-2-chloroethane or 1,2-dibromopropane, and therefore also other propanes should not differ. In the case of C KIE the scenario might be different as depending on the carbon position adjacent to the eliminated bromine substituent one can observe either larger or smaller isotope effect (~1.06 or 1.04 for the primary and secondary position, respectively). By predicting halogen binding isotope effects (BIEs) as well as computing interaction energy for each HLD-ligand complex the binding event preceding a chemical change in the active site has been characterized. The magnitude and direction of BIEs are discussed thoroughly by invoking the dynamics and the architecture of each active site, the strength of interactions with the first shell residues, and pointing to the dominating forces determining the binding of each of the studied ligands.