The work described in this thesis focused on the use of nuclear magnetic resonance spectroscopy (NMR) to study two classes of metallo enzymes - the Fe(II)- and 2oxoglutarate (2OG)-dependent dioxygenases and the metallo β-lactamases (MBLs). These enzymes are involved in clinically important biological processes, i.e. the hypoxic response and antimicrobial resistance, respectively. Both protein systems are interesting from an NMR perspective because they have dynamic regions involved in catalysis and ligand interactions. The work included mechanistic studies, protein-ligand interaction studies, and method development for inhibitor discovery. NMR was applied to study the human prolyl hydroxylase domain-containing protein 2 (PHD2), which is crucially involved in the chronic hypoxic response. The results reveal that binding of the C- and the N-terminus of the oxygen dependent degradation domains CODD and NODD, respectively, induce different interactions with PHD2. The substitution of a single amino acid, as occurs with PHD2 variants linked to erythrocytosis and breast cancer, can alter the selectivity of PHD2 towards its ODD substrates. Studies with the Trichoplax adhaerens PHD provide insights into the evolutionary substrate preference of the PHDs. Using <sup>13</sup>C-labelled peptidyl-substrates; NMR was applied to investigate proposed 'alternative' PHD2 substrates/interaction partners. The product release mechanism of PHD2 was investigated using NMR; the results reveal that the presence of 2OG strongly discriminates between the binding of CODD and hydroxylated CODD to PHD2. NMR was also applied to monitor PHD2 kinetics and inhibition. Competition and displacement assays were designed and applied to investigate PHD inhibitor binding modes. Comparative studies on the activities and selectivities of PHD inhibitors in clinical trials should aid in the work on the therapeutic manipulation of the natural hypoxic response. Protein-observe <sup>1</sup>9F-NMR was used to study the São Paolo MBL (SPM-1). The results provide new structural insights into SPM-1 catalysis and the requirements for inhibitor development. They also reveal that the hydrolysed β-amino acid products of MBL catalysis can bind to SPM-1. They illustrate the utility of <sup>19</sup>F-NMR for detecting metal chelation, which is not always readily tractable in studies on metallo enzyme inhibition, new binding modes, and stereoisomer binding/epimerisation in solution. The interaction of a cyclobutanone analogue, a broad-spectrum MBL inhibitor, with SPM-1 was investigated. A combination of <sup>1</sup>H, <sup>19</sup>F, <sup>13</sup>C-NMR and crystallographic analyses reveal that cyclobutanone binding may mimic formation of the oxyanion tetrahedral intermediate in β-lactam hydrolysis. The susceptibility of avibactam, the first clinically useful non-β-lactam β-lactamase inhibitor, to MBL-catalysed hydrolysis was studied. The results reveal that avibactam is not an MBL inhibitor and a poor substrate of most members of all three clinically relevant subclasses of MBLs. In some cases, avibactam undergoes slow hydrolysis in a process different from that observed with serine β-lactamases. Overall, the results illustrate the utility of NMR for studying dynamic aspects of enzyme catalysis and inhibitor binding.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:730154 |
| Date | January 2016 |
| Creators | Abboud, Martine |
| Contributors | Schofield, Christopher |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ora.ox.ac.uk/objects/uuid:a4aa5995-625a-4814-8c91-e0114c1e2004 |
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