University of Leicester
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Substrate binding : interactions in ascorbate peroxidase

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posted on 2014-12-15, 10:36 authored by Katherine Helen. Sharp
X-ray crystallography, kinetics, spectroscopy and directed evolution have been used to define substrate binding in recombinant soybean cystolic ascorbate peroxidase (rsAPX), which catalyses the hydrogen peroxide-dependent oxidation of ascorbate in plants.;The rsAPX crystal structure has been solved to 1.8 A (Chapter 2) and provides a useful comparison for the substrate bound structures. Nitric oxide and cyanide bound rsAPX crystal structures have also been solved to 2.0 A and are used to model the binding of hydrogen peroxide in APX (Chapter 2).;Ascorbate binding interactions are defined by the rsAPX/ascorbate crystal structure, which has been solved to 1.4 A (Chapter 3). This structure provides new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, and a mechanism for electron transfer from the substrate to the heme has been prepared (Chapter 3).;The crystal structure of the rsAPX/salicylhydroxamic acid (SHA) complex has been solved to 1.46 A (Chapter 4). Spectroscopic and kinetic techniques were used to show that guaiacol and SHA bind at the same site in APX. These data define the aromatic binding site in rsAPX and, along with the other crystal structures, have been used to propose a mechanism for proton transfer (Chapter 4).;A screen for enhanced aromatic oxidation has been developed, using guaiacol (colourless), which is oxidised to tetraguaiacol (red) by rsAPX (Chapter 5). The colour change and the fact it can be carried out on whole cells, allows many mutants to be assessed very efficiently. As a result it will be ideal for screening libraries of rsAPX mutants created by directed evolution.


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University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD



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