Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis.

Lindsay Mcgregor,Matthew P Blakeley,Edina Rosta,Tamás Földes,Roberto A Steiner,Nicolas Coquelle,Martine Moulin,Soi Bui

doi:10.1107/s2052252520013615

Lindsay Mcgregor, Matthew P Blakeley + Show 6 more

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https://doi.org/10.1107/s2052252520013615

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Abstract

Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to de-hydro-isourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hy-droxy-isourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the 'peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O-H⋯π interaction with 8AZA. The active site Lys10-Thr57 dyad features a charged Lys10-NH3 + side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1-HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.

Highlights

In many organisms, urate oxidase (UOX) is an essential enzyme that catalyses the O2-dependent degradation of uric acid (UA) to 5-hydroxyisourate (5HIU) (Kahn et al, 1997)
High-resolution X-ray crystallographic studies of the anaerobic complex of Aspergillus flavus UOX in complex with its natural substrate UA (Bui et al, 2014) as well as with several other similar scaffolds including the 8-azaxanthine (8AZA) inhibitor employed in this study [Fig. 1(b)] (Colloc’h et al, 1997; Gabison et al, 2010; Retailleau et al, 2004), reveal that ligands bind at the interface between two protomers of the $137 kDa UOX tetramer stabilized by several hydrogen bonds with the additional contribution of a – stacking interaction [Figs. 1(c) and 1(d)]
We have integrated high-resolution room-temperature neutron/X-ray crystallographic analysis with biomolecular simulations to study co-factor-independent UOX in complex with its competitive inhibitor 8AZA in the presence of the catalytic water molecule W1 bound in the peroxo hole

Summary

Introduction

Urate oxidase (UOX) is an essential enzyme that catalyses the O2-dependent degradation of uric acid (UA) to 5-hydroxyisourate (5HIU) (Kahn et al, 1997). UOX-mediated degradation of UA follows two sequential steps: (1) an initial oxidation step whereby UA reacts with O2 to yield dehydroisourate (DHU) via a 5peroxoisourate (5PIU) intermediate and (2) a hydration step in which DHU is hydroxylated to 5HIU [Fig. 1(a)] (Kahn, 1999; Wei et al, 2017). Small negative ions like cyanide and chloride have been visualized in the peroxo hole in the presence of intact UA under aerobic conditions (Gabison et al, 2008, 2010; Oksanen et al, 2014) offering structural support for their behaviour as competitive inhibitors

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