Carnitine is an essential metabolite in the human body. It carries out several roles in human metabolism, including that in fatty acid metabolism. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate dependent oxygenase, which catalyses the final step of carnitine biosynthesis, i.e. hydroxylation of γ-butyrobetaine (GBB) to carnitine. Inhibition of BBOX has potential in the treatment for cardiovascular diseases. The work described in this thesis focussed on mechanistic and inhibition aspects of BBOX catalysis. Firstly, a set of analytical tools for BBOX activity measurements was developed. The synthesis of fluorinated substrate analogues provided the basis for development of two assays for use in vitro with the isolated protein and in lysates, with detection by fluorescence or <sup>19</sup>F NMR, respectively. Furthermore, the use of <sup>19</sup>F NMR to monitor protein-ligand interactions was exemplified with the work on metallo-β-lactamases. The developed fluoride-release assay was then used to screen a library of small molecules and led to recognition of scaffolds with potential applications as inhibitors. Further structure-activity relationship studies led to the identification of potent BBOX inhibitors, which were then evaluated for their activity in cells. The crystal structure of human BBOX with one of the lead inhibitors revealed that BBOX can undergo significant conformational changes, involving a movement of an active site loop. BBOX conformational flexibility may have a role in the GBB mediated substrate inhibition observed both with isolated protein and in cells. In addition to the mechanistic and functional studies, the potential of BBOX as a biocatalytic tool was examined. BBOX has been shown to catalyse a hydroxylation of the symmetrical dialkyl piperidine carboxylic acids, leading to formation of up to three stereocentres in one reaction. In the last part of this work properties of human BBOX were compared to BBOX from Pseudomonas sp. AK1, revealing differences in kinetic behaviour and substrate specificity. Novel substrates for bacterial BBOX were identified. Pseudomonas sp AK1 BBOX was shown to hydroxylate amino acid analogues leading to formation of 1,2-amino alcohols.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:669830 |
| Date | January 2014 |
| Creators | Rydzik, Anna Maria |
| Contributors | Schofield, Christopher J. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:cccc1dd1-c063-4820-8ab1-58d698b04616 |
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