Mechanism-Based Discovery of Novel Substrates of Haloalkane Dehalogenases Using in Silico Screening

Substrate specificity is a key feature of enzymes determining their applicability in biomaterials and biotechnologies. Experimental testing of activities with novel substrates is a time-consuming and inefficient process, typically resulting in many failures. Here, we present an experimentally validated in silico method for the discovery of novel substrates of enzymes with a known reaction mechanism. The method was developed for a model system of biotechnologically relevant enzymes, haloalkane dehalogenases. On the basis of the parametrization of six different haloalkane dehalogenases with 30 halogenated substrates, mechanism-based geometric criteria for reactivity approximation were defined. These criteria were subsequently applied to the previously experimentally uncharacterized haloalkane dehalogenase DmmA. The enzyme was computationally screened against 41,366 compounds, yielding 548 structurally unique compounds as potential substrates. Eight out of 16 experimentally tested top-ranking compounds were ...