New insight into the molecular mechanism of the trehalose effect on urate oxidase stability

Abstract

Urate oxidase (EC 1.7.3.3) is a key enzyme in the purine metabolism which is applied in the treatment of gout and also, as a diagnostic reagent for uric acid detection. In the current study, the trehalose (TRE) effects as an additive on the structural stability and function of uricase were investigated. For recombinant expression of UOX in E. coli BL21 cells, firstly the coding sequence was subcloned into the pET-28a vector and after induction with IPTG, the recombinant UOX was purified by affinity chromatography using a Ni-NTA agarose column. To specify the trehalose effects on the urate oxidase (UOX) structure, optimum pH, optimum temperature, kinetic and thermodynamic parameters and also, the intrinsic fluorescence of UOX in the absence and presence of trehalose were examined. The UOX half-life is 24.32 min at 40 °C, whereas the UOX-TRE has a higher half-life (32.09 min) at this temperature. Generally, our findings confirm that trehalose has a protective effect on the enzyme structure. Optimum pH and temperature were 9 and 25 °C, respectively for both the naked and treated enzymes and their activity retained 42.18 and 64.80%, respectively after 48 h of incubation at room temperature. Also, theoretical results indicate that the random coil of the enzyme was converted to α-helix and β-sheet in the presence of trehalose which may preserve the integrity of the active site of the enzyme and increased the enzymatic activity. The MD simulation results indicated greater stability of the uricase structure in the presence of trehalose.

Communicated by Ramaswamy H. Sarma

Keywords:

• Urate oxidase
• trehalose
• thermal stability
• molecular dynamics simulations
• kinetic and thermodynamic parameters

Disclosure statement

The authors declare no competing financial interest.

Funding

We would like to thank the Graduate University of Advanced Technology for the support of this work.