Investigating the molecular basis of organohalide respiration

  • Carolina Quezada Bustos

    Student thesis: Unknown


    During the last century, pollution as a consequence of industrialization and urban growth is threatening the global ecosystem. Some of the most important environmental pollutants are halogenated organic compounds (organohalides), xenobiotic molecules that are often highly toxic and recalcitrant. Due to their physicochemical characteristics, organohalides tend to accumulate in anoxic ecosystems. Nevertheless, organohalides are not only anthropogenic in origin, but part of a natural halogen cycle and produced by both abiotic and biological processes. Certain microorganisms have developed the capacity to use organohalides as terminal electron acceptors, in a process termed organohalide respiration. As organohalide reduction leads to dehalogenation, it has potential to be used in bioremediation of contaminated sites. However, until recently relatively little was known about the key enzyme supporting this process, the reductive dehalogenase (RdhA) and its transcriptional regulation. This project aimed to characterise at the biochemical and structural level a presentative RdhA enzyme and one of the associated transcription factors of the MarR-type (RdhR). Here, the crystal structure of a catabolic reductive dehalogenase from Nitratireductor pacificus pht-3B (NpRdhA) that catalyses the debromination of 3,5-dichloro-4-hydroxybenzoic acid, a herbicide derivate, is reported. Active NpRdhA was heterologously expressed and aerobically purified from Bacillus megaterium. The direct interaction between the cobalamin cobalt and the substrate halogen that underpins catalysis is shown by combining structure determination with EPR spectroscopy and simulation. In contrast to other cobalamin-dependent subfamilies, here it is proposed that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. It was also tested whether the standard bacterial host E. coli can be used for rdhA heterologous expression when B12 uptake is enhanced. This strategy worked well for the catabolic NpRdhA, leading to the production of active holo-enzyme in vivo, but not for the respiratory reductive dehalogenases tested. In addition, crystal structures of Dehalococcoides mccartyi strain CBDB1 RdhRCbdbA1625 are presented, both ligand free and in complex with three dichlorophenolic ligands. These ligand-bound complexes revealed a single organohalide-binding site that contains three distinct chlorine-binding pockets. A fragment-merging type approach yielded identification of 2,3,4-trichlorophenol as a tight binding ligand for this regulator, suggesting it is a possible substrate for the associated RdhA9 (CbdbA1624). These findings have provided the first insights into the RdhA and RdhR structure-function relationships, and will guide protein-engineering efforts towards bioremediation or biosensing applications.
    Date of Award1 Aug 2016
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorDavid Leys (Supervisor)


    • Organohalide respiration, crystal structure, reductive dehalogenase, B12, MarR regulator

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