Organohalide pollutant detoxification using engineered reductive dehalogenases

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The reductive dehalogenases (RDases) are key enzymes in organohalide respiration, which is performed by a unique subset of bacteria. The RDases are cobalamin-dependent and able to selectively cleave carbon-halogen bonds through reduction. As a result, RDase containing organisms have been targeted as a potential bioremediation tool for the clean-up of contaminated anaerobic sites where organohalides have accumulated, often due to improper disposal. The RDases can be split into two classes: i) the canonical respiratory dehalogenases, which use a halogenated compound as a final electron acceptor during organohalide respiration and ii) the catabolic respiratory dehalogenases that occur in the catabolic pathways of non-organohalide respiring bacteria [3]. The catabolic RDase enzymes tend to be oxygen-tolerant, suggesting these are more desirable targets for future mechanistic studies and bioremediation applications. A range of mechanisms have been proposed including long range versus short range/inner sphere mechanisms on the basis of substrate/ligand complex crystal structures. It thus remains unclear what are the fundamental drivers of appropriate substrate positioning and B12 reduction. Making use of a range of catabolic reductive dehalogenases we will determine what governs substrate specificity and explore the scope to rationally repurpose this. We will establish both the mechanism of B12-mediated substrate reduction (using in crystallo approaches) and NAD(P)H mediated B12-reduction (using spectroscopic approaches) to provide a complete mechanistic understanding of catalysis. We will use fundamental understanding gained at Manchester to guide in vivo bioremediation of environmentally relevant organohalides at Toronto. The more fundamental aspects of the study will be carried out at Manchester in the MIB taking advantage of the local strength in enzymology, protein engineering and biotechnology. The in vivo bioremediation studies will be carried out in Toronta at BioZone taking advantage of the local expertise in chemical engineering. We propose the student will spend a ~12 month placement in the 3rd year at the Edwards lab in BioZone. The project fits both within the world class bioscience as well as industrial biotechnology areas.