(185ac) Role of Intersubunit Hydrogen Bond to Urate Oxidase Stability: Molecular Simulation and Experimental Validation

Urate oxidase (UOX, EC 1.7.3.3), a tetrameric ?Tunnelling-fold? enzyme for the conversion of uric acid, is effective for the treatment of gout and hyperuricaemia associated with tumor lysis syndrome. The inherent poor stability of UOX to temperature, proteolysis and acidic environment is known to limit its efficacy while the deactivation mechanism is poorly understood, particularly at molecular level.

This presentation started with a molecular dynamic simulation of aqueous UOX, which illustrated that the intersubunit hydrogen bond was essential for maintaining the native conformation of UOX. Also, we have proved the stability improvement of UOX can be achieved by superseding water molecules surrounding UOX surface with solvents of weak compete for the intersubunit H-bond.

Experimental validation was performed using SEC, circular dichroism and florescence spectroscopy to monitor the conformational changes of UOX. It was shown that the secondary and tertiary structure unfolding instead of quaternary structure dissociation occurred during inactivation process of UOX. This demonstrated the crucial role of H-bonding at subunit-subunit interface, as illustrated by the molecular simulation. Furthermore, as predicted by the in silico experiment described above, the presence of methanol and DMSO in aqueous UOX enhanced the stability of UOX and stabilized its tertiary structure as proven by activity assay and fluorescence spectrum results, which also supported the stabilization mechanism by MD simulation.

Chemical modification of UOX was then attempted via two-step aqueous in situ polymerization and fabricated UOX nanogel with a diameter of 20-40nm. Compared to its native counterpart, UOX nanogel exhibited a significantly enhanced stability against low pH, high temperature, and more importantly, proteolysis, proved by activity assay, structural change and integrity. The proposed method is helpful for the further development of UOX formulation in drug delivery system.