The evolution of microorganisms over millions of years has led to an impressive adaptability regarding the utilisation of different environmental conditions. The identification of bacterial species with the fascinating features to use cobalamin-dependent metalloenzymes to (i) extract energy from halogenated organic compounds (organohalides) and (ii) transfer methyl groups from lignin breakdowns into central carbon pathways, are examples for this adaptability. The biochemical study of these two cobalamin-dependent enyzmes is the topic of this PhD project.For the extraction of growth energy, organohalides serve as terminal electron acceptorsand are reductively dehalogenated in a respiratory manner termed organohalide respiration. Reductive dehalogenases, the key enzymes in organohalide respiration, catalyse the chemical cleavage between the halogen substituent and the carbon moiety. They use cobalamin and two Fe-S clusters as cofactors and constitute a new and distinct class of cobalamin-dependent enzymes. Their three-dimensional structure and the mechanism of catalysis are unknown, because their hydrophobicity and oxygen sensitivity have hampered their biochemical investigation. Here, a novel purification technology in Escherichia coli for the reductive dehalogenase PceA from Dehalobacter restrictus has been developed, accompanied by methods that allow the in vitro reconstitution of PceA with both cofactors, cobalamin and Fe-S clusters. It has been demonstrated that the soluble expression of PceA is dependent on the covalent fusion of the enzyme to a trigger factor chaperone. Based on these findings, the PceA specific trigger factor PceT has been studied biochemically, resulting in its successful crystallisation. The established protocols for PceA and PceT are transferable to other members of their respective families, which will therefore allow detailed studies of reductive dehalogenases and their associated chaperones in the future.In addition to reductive dehalogenases, organohalide respiring bacteria contain anothercobalamin-dependent enzyme system, termed O-demethylase, which is involved in the carbon metabolism of different anaerobic bacteria. O-demethylases are three-component enzyme systems that transfer methyl groups from aromatic methyl ethers totetrahydrofolate via methylcobalamin intermediates. The different cofactors (substrate,cobalamin and tetrahydrofolate), bind to either of the three individual proteins involvedin O-demethylation. It has been speculated that the same or similar halogenated aromatic molecules are substrates for both organohalide respiration and O-demethylation in the same bacteria. In order to test this proposal, a O-demethylase from Desulfitobacterium hafniense DCB-2 has been studied using X-ray crystallography and biochemistry. As a result, the first crystal structures of the cobalamin-binding protein in complex with cobalamin, and of the methyl acceptor protein in complex with substrate (tetrahydrofolate) and product (methyltetrahydrofolate) from a O-demethylase have been solved toresolutions of 1.5 A, 1.8 A and 1.6 A, respectively. The crystal structures, in combinationwith spectroscopic and biophysical analyses, have led to a proposed mechanism forthe catalysed methyl transfer reaction from methylcobalamin to tetrahydrofolate.
|Date of Award
|31 Dec 2013
- The University of Manchester
|David Leys (Supervisor) & Nigel Scrutton (Supervisor)