Biochemical basis of organohalide degradation

  • Samantha Gaytan Mondragon

Student thesis: Phd


Organohalide respiring bacteria (OHRBs) can use halogenated compounds as terminal electron acceptors, leading to organohalide reduction and degradation. Organohalides are naturally produced as part of the halogen biogeochemical cycles, but anthropogenically introduced compounds are now considered major environmental pollutants. All OHRBs rely on reductive dehalogenases (RdhAs), a subfamily of B12-dependent enzymes, that catalyse the final step of the organohalide respiration. However, RdhA enzymes are not exclusively found in anaerobic OHRBs, and a subset of RdhAs are part of catabolic pathways in marine Proteobacteria. These enzymes catalyse the reduction of organohalides to allow the complete degradation or reutilisation of the carbon backbone. A complete understanding of the structure, function, and regulation of the RdhAs is desirable for the application of reductive dehalogenation in the bioremediation of anaerobic polluted sites. Here we present the initial biochemical characterisation of a catabolic RdhA that consists of a natural fusion between the dehalogenase B12/Fe-S domains and an additional C-terminal reductase domain, similar to the iron-sulfur (Fe-S) flavoprotein phthalate dioxygenase reductase (PDR). The RdhA-PDR fusion renders the enzyme self-sufficient in terms of coupling NAD(P)H oxidation to organohalide reduction and are thus referred to as self-sufficient RdhAs (ssRdhAs). To achieve functional heterologous expression of ssRdhAs, we used the xylose-inducible B. megaterium and the standard E. coli system optimised with a B12-uptake system (BtuB). Although the cofactor incorporation and protein yield remain a problem despite extensive screening of a range of conditions, we could obtain in vivo and in vitro activity with ortho-halogenated phenols. Furthermore, we confirmed that the PDR-like domain allows the intramolecular transfer of electrons from NAD(P)H to the active site of cobalamin without the need for an external reductase system to provide electrons. Given the initial challenges of producing soluble and active full-length enzymes, we also expressed the isolated PDR-like reductase domain and demonstrated it can support dehalogenation by channelling electrons to the dehalogenase active site when assayed in a one-pot reaction. Additionally, to test these results, we designed and produced an artificial fusion enzyme linking the PDR-like reductase domain with the previously characterised non-self-sufficient NpRdhA. Unfortunately, no consistent dehalogenation of the substrate 3,5-Bromo-4-hydroxybenzoic acid was obtained. To address the regulation of the RdhAs, we completed the structural and functional characterisation of the MarR-type transcriptional regulator RdhRCbdb1625, previously studied in terms of ligand specificity towards dichlorinated phenols. In this work we present the crystal structure of RdhRCbdb1625, in complex with a 1,2,3-tricholorophenol, providing a rationale for the distinctive preference for specific halogenation substitution patterns. We also demonstrate the effect of the tight-binding ligands on the RdhRCbdb1625 DNA-binding affinity, confirming its putative role as a transcriptional repressor.
Date of Award1 Aug 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorDavid Leys (Supervisor) & Jonathan Waltho (Supervisor)

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