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Title: Investigating the role of calcium ions in Escherichia coli Amine Oxidase
Author: Pirrat, Pascale
ISNI:       0000 0001 3491 8889
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2007
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In copper amine oxidases the role of the buried active site copper has been extensively studied, while the functions of the peripheral metal ions, assigned as calcium according to crystallographic data, have-largely been ignored. One peripheral metal site slightly buried and called for clarity the 'buried site', is structurally coupled to the copper and two of its ligands are strictly conserved. In Escherichia coli amine oxidase (ECAO), as in Pichia pastoris Iysyl oxidase, Bovine Serum amine oxidase and Vascular adhesion protein, another peripheral metal site, the 'surface site', which is rather poorly conserved, is found near the surface of each subunit. Solution studies have been performed showing that ECAO activity is reduced upon EDTA treatment but can be recovered by addition of calcium and other metal ions (S~+, Mn2+, Mg2+). These studies together with crystallographic data of various ECAO derivatives (EDTA-treated , Strontium soaked and Xenonexposed- ECAO) showed that (i) the EDTA treatment displaces both calcium ions, the buried one being responsible for the reduction of activity upon EDTA treatment (ii) both peripheral sites are normally occupied by calcium ions but can be replaced by other metal ions, with the surface site displaying a lower affinity for calcium than the buried site, (iii) there are some preferred routes for O2 substrate entry, one is also the route for the amine substrate entry, the other one is structurally connected with the peripheral sites. Site-directed mutagenesis studies have also been conducted involving the ligands of both peripheral metals. While all surface site mutants expressed well, none of the buried mutants expressed a full-size ECAO unless the growth medium was supplemented with calcium. Amine oxidase activity is mediated by the redox active cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), which is the result of the post-translational modification of a tyrosine residue. In all surface site mutants studied so far by crystallographic and spectroscopic means, TPQ synthesis was prevented or reduced. The possible mechanisms behind these effects are discussed. Overall this work proposes a role in enhancing activity for the buried site and a surprising but critical role for the surface site in supporting TPQ biogenesis. Both sites could facilitate the folding process.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available