Characterisation of two clinical mutations of Xanthine oxidoreductase related to hyperuricaemia

Abstract

Xanthine oxidoreductase (XOR) is a molybdoflavin enzyme and a homodimer with a mass of 290 kDa. In mammals, XOR exists in two interconvertible forms: Xanthine Dehydrogenase (XDH) and Xanthine Oxidase (XO). XOR catalyses the final steps of purine catabolism, converting hypoxanthine to xanthine and subsequently to uric acid. These two reactions can generate reactive oxygen species (ROS), specifically hydrogen peroxide and superoxide anion radicals. XOR has been associated with various health conditions, including hyperuricemia, oxidative stress, and hypertension. ROS generated by XOR contribute to endothelial dysfunction, with increased ROS levels reacting with nitric oxide (NO) to form peroxynitrite radicals, exacerbating hypertension. Different variants have also been linked with increased XOR activity as well as higher risk of diseases, such as hyperuricemia and hypertension. In this project, the hXOR wildtype (WT) and two known clinical variants identified in a Maltese study by the MAMI study, I646V and I703V, as well as a novel double mutant (I646V-I703V) also identified in the Maltese population, were characterised. Apart from the clinical variants, the I646A mutation was also constructed to characterise to assess the effect of the substitution of a large non-polar branched amino acid such as isoleucine with a smaller one. The aim of this project was to determine the effect these amino acid changes have on the hXOR protein. Transformed TP1000 E.coli cells harbouring either the pTrc-HisXOR WT or the variants were grown under aerobic conditions without and with heat shock. Purification was performed using immobilised metal affinity chromatography. The purified proteins were characterised using SDS-PAGE, Native-PAGE, Blue-Native gel, and Dynamic Light Scattering (DLS) for size and oligomeric state. The enzymatic activity was evaluated using the uric acid spectrophotometric assay and the Nitro Blue Tetrazolium (NBT) assay. Secondary structure, thermal stability (melting temperature), and amyloid fibril formation were assessed using circular dichroism, DLS, and the Thioflavin T (ThT) assay, respectively. The amyloidogenic potential was further investigated using the ZipperDB server. All variants exhibited significantly higher enzymatic activities compared to the WT hXOR sample, with the I646A variant showing the highest activity (9-fold greater than WT). Analysis of DLS data confirmed that the native proteins of all mutants predominantly occur in the active native dimer conformation. Conversely, the WT hXOR exhibited the lowest activity and formed the largest oligomeric structures. The WT hXOR formed amyloid fibrils in the ThT assay, supported by the identification of amyloidogenic stretches using ZipperDB. Most variants did not show fibrillization, specifically I703V, I646A, I646V+HS, I646V-I703V+HS, and I646A+HS. The substitution of Ile646 with Valine or Alanine (I646V, I646A) significantly reduced the amyloidogenic propensity while increased substantially the enzymatic activity. I646V-I703V+HS also exhibited high enzymatic activity and displayed no fibril formation. These findings show that while WT hXOR activity is dampened by fibril formation, the clinical variants remain hyperactive and stable. This high activity of hXOR may result in significant ROS production and hyperuricemia potentially leading to various health conditions, such as hypertension.