Neutron diffraction studies of perdeuterated urate oxidase complexes

Abstract

Urate Oxidase (UOX) is the archetypal cofactor-free oxidase and has been the subject of several biochemical studies over the last 70 years. This enzyme plays a role in purine recycling, facilitating the O2-mediated degradation of uric acid to 5-hydroxyisourate. The latter compound is then further degraded to the more soluble allantoin. However, there are significant thermodynamic and kinetic barriers associated with O2-activation, and it is not yet completely understood how UOX is able to function in the absence of a redox-active metal or organic cofactor. The main aim of this PhD project was to advance our mechanistic understanding of UOX catalysis by investigating the structure of UOX complexes using neutron macromolecular crystallography (NMX). Compared to the more popular technique of X-ray crystallography (MX), the main strength of NMX is that it allows the direct visualization of hydrogen atoms (typically in the form of deuterium atoms). As a correct understanding of enzymatic catalysis often relies on the proper identification of protonation states at different stages of the reaction, NMX is a very valuable tool in mechanistic enzymology studies. This thesis is divided in six chapters. In the introductory Chapter 1, the subject of O2 reactions in biology is presented, with reference to the challenges associated with O2 activation. A historical overview of the current literature available on UOX is provided, including its clinical relevance, in addition to its structural and kinetic studies. Finally, the remaining mechanistic challenges associated with UOX are discussed. Chapter 2 briefly describes the main methodologies used in this work. In Chapter 3, I present the recombinant production, purification, and characterisation of perdeuterated UOX (DUOX) that was employed for all NMX/MX experiments described in this thesis. As the use of a perdeuterated sample presents significant advantages over the use of a H/D-exchanged one in terms of NMX data quality and maps interpretation, such an approach is a substantial improvement over previous NMX studies carried out on UOX by other authors. The joint NMX/MX refinement of the room-temperature complex between DUOX and the inhibitor 8-azaxanthine (AZA) is described in Chapter 4. This structure determines the 2 protonation states of the proposed catalytic triad, Thr57*-Lys10*-His256, and the relationships between this triad and the active site solvent network are determined. In Chapter 5, I present the room-temperature NMX structure of DUOX in complex with 9-methyl-5-peroxyisourate (5-PMUA). The latter compound is trapped in the active site after co-crystallisation of DUOX with 9-methyl uric acid (9-MUA) under aerobic conditions, allowing the binding of O2. From this structure, the trapped peroxide species is found to be negatively charged and is stabilized by the hydroxyl group of active site residue Thr57*. In addition, a proton-sharing relationship between residues Lys10* and His256 of the catalytic triad is established. NMX was employed to obtain information on the protonation states of UOX complexes in two different environments: in the presence of an inhibitor that cannot be oxidised, and the trapped peroxide intermediate. The structures that are presented in this thesis are discussed with reference to the current knowledge of the UOX mechanism of action, and provide an exciting platform for future work into the structure of reactive intermediates in UOX catalysis.