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

Cofactor-independent urate oxidase (UOX) is an similar to 137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O-2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O-2 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 degrees with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O-H center dot center dot center dot pi 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 pi system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 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/O-2 exchange during catalysis.

Automated summary

This study investigates the hydration step of urate oxidase reaction using high-resolution neutron/X-ray crystallographic analysis and molecular dynamics simulations. The catalytic water molecule W1 plays a crucial role in the reaction, stabilized by interactions with specific residues and the inhibitor. Conformational heterogeneity of Asn254 is proposed as an active mechanism to facilitate W1/O-2 exchange during catalysis.