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

doi:10.1107/S2052252520013615

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

IUCrJ. 2021 Jan 1;8(Pt 1):46-59. doi: 10.1107/S2052252520013615.

Authors

Lindsay McGregor^{1

2}, Tamás Földes^{3

4}, Soi Bui¹, Martine Moulin⁵, Nicolas Coquelle², Matthew P Blakeley², Edina Rosta^{3

4}, Roberto A Steiner¹

Affiliations

¹ Randall Centre for Cell and Molecular Biophysics, King's College London, London SE1 1UL, United Kingdom.
² Large Scale Structures Group, Institut Laue-Langevin, 71 avenue des Martyrs, 38042 Cedex 9, Grenoble, France.
³ Department of Chemistry, King's College London, London SE1 1DB, United Kingdom.
⁴ Department of Physics and Astronomy, University College, London WC1E 6BT, United Kingdom.
⁵ Life Sciences Group, Institut Laue-Langevin, 71 avenue des Martyrs, 38042 Cedex 9, Grenoble, France.

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 Thr57^OG1, while the Thr57^OG1-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 DHU^C5 with Thr57^HG1 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.

Keywords: biomolecular simulations; cofactor-independent oxidase; neutron/X-ray diffraction; protein perdeuteration; urate oxidase.

Grants and funding

This work was funded by Biotechnology and Biological Sciences Research Council grant BB/P000169/1 to Roberto Steiner and Edina Rosta. French Infrastructure for Integrated Structural Biology grant . Communauté Université Grenoble Alpes grant . King’s College London grant . Engineering and Physical Sciences Research Council grant EP/R013012/1 to Edina Rosta. European Research Council grant 757850 to Edina Rosta.