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Title: Investigating intersubunit communications and the oxidative half-reaction in copper amine oxidases
Author: Gaule, Thembaninkosi Grace
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Copper amine oxidases (CuAOs) are ubiquitous, copper containing, homodimeric enzymes that possess a protein derived cofactor 2, 4, 5- trihydroxyphenylalanine quinone (TPQ). Crystal structures of CuAOs from a range of organisms reveal quaternary structure conservation with two long ~hairpins (arm I and arm II) and peripheral metal sites. The arms protrude from one monomer to embrace the other. The functional roles of these ~hairpins are not clearly understood although they are implicated in structural stability of the dimer. Arm I consists of residues that form a highly conserved hydrogen bond network with residues in close proximity to the active site. This interaction network could potentially be involved in inter-subunit communication as co-operativity has been proposed for some CuAOs. To investigate the extent of co-operativity and the structural basis of inter-site communication we have developed an expression system able to produce heterodimeric forms of A. globiform;s GuAO. By co-expressing genes encoding the Wild-type and an active site mutant, we have demonstrated effective dual tag affinity purification of heterodimers. Escherichia coli amine oxidase (EGAO) contains two peripheral metal sites; the buried metal site and the surface metal site. The buried site, which is structurally connected to the active site copper via two a-strands, is highly conserved in seven out of the eight structurally determined GuAOs. In contrast, the surface metal site is poorly conserved. Site directed mutagenesis studies have been used to investigate the roles of the peripheral metal sites in ECAO. The buried site was found to be essential for the synthesis and folding of the protein. In contrast mutation of the surface metal site did not affect protein expression levels and good yields of well folded protein could be obtained. However, mutation of the surface metal site ligand glutamate 573, which prevents the coordination of the calcium ion, resulted in a dramatic decrease in the catalytic activity. Introducing a second mutation at a different site resulted in partial rescue of catalytic activity. How this occurs is not clear, however a combination of techniques such as UV-vis spectroscopy, Terahertz time domain spectroscopy, kinetics, X-ray crystallography and electrophoretic studies have provided some insight into the mechanisms of EGAO.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available