7A0L

Joint neutron/X-ray room temperature structure of perdeuterated Aspergillus flavus urate oxidase in complex with the 8-azaxanthine inhibitor and catalytic water bound in the peroxo hole

Summary for 7A0L

• Entry DOI: 10.2210/pdb7a0l/pdb
• Descriptor: Uricase, 8-AZAXANTHINE, SODIUM ION, ... (4 entities in total)
• Functional Keywords: cofactor-free oxidase, inhibitor, catalytic water, dioxygen binding, oxidoreductase
• Biological source: Aspergillus flavus
• Total number of polymer chains: 1
• Total formula weight: 34464.83

Authors

McGregor, L.,Bui, S.,Blakeley, M.P.,Steiner, R.A. (deposition date: 2020-08-09, release date: 2020-12-09, Last modification date: 2024-11-20)

Primary citation

McGregor, L.,Foldes, T.,Bui, S.,Moulin, M.,Coquelle, N.,Blakeley, M.P.,Rosta, E.,Steiner, R.A.
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, 8:46-59, 2021

PubMed 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 O 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 O 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 UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral HO, 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-NH side chain engaged in a strong hydrogen bond with Thr57, while the Thr57 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 with Thr57 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/O exchange during catalysis.

PubMed: 33520242
DOI: 10.1107/S2052252520013615

Experimental method

NEUTRON DIFFRACTION (2.1 Å)
X-RAY DIFFRACTION (1.33 Å)