Unravelling the mechanisms of xanthine oxidoreductase chain formation : insights into protein structure and function

Abstract

Xanthine oxidoreductase (XOR) catalyses the last two steps of purine catabolism, converting hypoxanthine to xanthine and subsequently to uric acid. From these reactions, hydrogen peroxide and superoxide anion radicals can be generated. Increased activity of XOR may result in hyperuricemia due to higher production of uric acid and oxidative stress from increased generation of reactive oxygen species (ROS). Hyperuricemia is associated with cardiovascular diseases, including stroke, and hypertension. Hypertension is linked to the depletion of nitric oxide by ROS and the subsequent production of peroxynitrite radicals. Various mutations have also been associated with high XOR activity as well as a higher risk of different conditions, such as hypertension, hyperuricemia, and xanthinuria. From previous studies, it was also shown that XOR can form filaments that may have a role in its enzymatic activity. In this project, the hXOR wildtype (WT) and one clinical mutation identified in a Maltese study, I646V, were characterised. The aim of the project was to determine of wild type hXOR and the I646V assembled into filaments formation and whether the filaments are enzymatically active. Transformed TP1000 E. coli cells harbouring either the pTrcHis-hXOR WT or I646V variant were grown under aerobic and anaerobic conditions in order to compare the filament formation and enzymatic activity. This approach aimed to elucidate the enzymatic role in physiological ischaemic conditions. Some samples were also subjected to heat shock to enhance the folding and activity of hXOR. Purification was carried out using immobilized metal affinity chromatography, with purity and yield assessed by SDS-PAGE. Filament presence was confirmed via Native-PAGE. Additionally, hXOR activity was evaluated using the uric acid spectrophotometric assay and the Nitro Blue Tetrazolium (NBT) assay. The secondary structure and melting temperature were analysed using circular dichroism. Production of XOR under anaerobic conditions resulted in a higher enzymatic activity for both the WT and I646V variant. For the samples produced under aerobic conditions, the I646V variant had the highest activity but had the lowest yield and stability. On the other hand, hXOR WT grown under anaerobic conditions was the most active out of all conditions tested and had the highest stability. Filament formation was observed in most conditions, with anaerobic growth conditions seemingly favouring filament formation for both WT and mutant. Moreover, the filaments formed retained activity as shown in the NBT assays. These findings are particularly important as filament formation might modulate the enzymatic activity of hXOR. Additionally, the observed increase in hXOR activity under anaerobic conditions is noteworthy, as physiologically, hXOR can be produced under hypoxic and ischemic conditions. The high activity of hXOR produced in such environments may result in significant ROS production, potentially leading to various adverse health conditions.