McGregor, L;
Földes, T;
Bui, S;
Moulin, M;
Coquelle, N;
Blakeley, MP;
Rosta, E;
(2021)
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
, 8
(Part1)
pp. 46-59.
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 dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (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.
| Type: | Article |
|---|---|
| Title: | 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 |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1107/S2052252520013615 |
| Publisher version: | http://dx.doi.org/10.1107/S2052252520013615 |
| Language: | English |
| Additional information: | This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Physics and Astronomy |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10119280 |
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