The Role of Gln269Leu Mutation on the Thermostability and Structure of Uricase from Aspergillus Flavus

Aspergillus flavus Urate oxidase (AFUOX) is promising for potential therapeutic applications, particularly in gout treatment. However, the enzyme's low thermostability and solubility limit its efficacy. A targeted mutation, substituting Gln with Leu at position 269 (Q269L) has been proposed to enhance its stability. The turnover number, catalytic efficiency, and specific activity of Q269L were 3.7 (s), 53.2 (mM. s), and 3.926 U/mg, respectively. In comparison, for the wild type, these were 3.1 (S), 35.1 (mM. s), and 4.018 U/mg, respectively. Notably, the wild type exhibited maximum activity at pH 9 and 25 °C, whereas the activity of Q269L was obtained at pH 9.5 and 30 °C. Furthermore, the half-life of Q269L at 40 °C is significantly longer (85.55 min) compared to the wild-type (49.85 min). The thermodynamic parameters ΔH, ΔS, and ΔG at 40 °C for Q269L were 60.9 kJ.mol, -276 J.mol, and 147.3 kJ.mol, respectively. Intrinsic fluorescence reductions and ANS fluorescence increases suggest that tryptophan resides in a polar environment with augmented hydrophobic pockets. FTIR analysis of Q269L reveals a decrease in β-sheet and an increase in α-helix structures, supporting molecular dynamics simulations. Collectively, MD and experimental results underscore Q269L's enhanced thermostability and localized structural alterations, advancing AFUOX's therapeutic potential.