Structural and biochemical analyses of human and sheep xanthine oxidoreductase

Abstract

Human recombinant xanthine oxidoreductase (hXOR) and ovine XOR were purified to homogeneity and characterised using various biophysical techniques. XOR catalyses the last two steps of purine degradation to produce uric acid using either NAD+ or oxygen as the electron acceptor. When oxygen is used, the reactive oxygen species (ROS) hydrogen peroxide and superoxide are produced. Various studies have shown that ROS are implicated in a number of pathological conditions including cardiovascular diseases. Transmission electron microscopy of both hXOR and ovine XOR revealed that these proteins form soluble filaments under physiological conditions and that the majority of these filaments are composed of two interacting dimers. A hypothesis is proposed which describes the assembly of the filaments in relation to their catalytic function. This may be verified by further analysis of the cryo electron microscopy (cryoEM) data of both the dimer and filamentous hXOR that has already been collected. The structure of hXOR, determined by X-ray crystallography, yielded a resolution of 3.95 脜 with a space group of P 31 2 1. The optimised expression system was used to analyse the activity of three clinicalrelevant hXOR mutants (G172R, A932T, N1109T), which have been implicated in hypertension. Only hXOR[A932T] exhibited altered activity, increasing three-fold with respect to the wild type. In silico structural studies indicate that this may be due to the additional interactions that T932 forms with neighbouring residues, absent in the wild type. T932 was found to be in a helix connected to the molybdenum active site and the mutation itself might shift the amino acids in the catalytic site, resulting into this increase in activity. Moreover, the inhibitory action of two recently published XOR inhibitors, 4- [(Z)-(6-hydroxy-3-oxo-1-benzofuran-2(3H)-ylidene)methyl]benzoic acid (inhibitor A) and (2E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (inhibitor B) was evaluated with IC50 values of 0.648 渭M and 45.5 渭M respectively. The mode of interaction of these inhibitors to hXOR was determined via docking experiments that showed that both inhibitors blocked the solvent funnel leading to the molybdenum centre. Inhibitor A, which had better inhibitory properties and had some structural homogeneity to purines, also formed stronger interactions with neighbouring residues. The formation of hXOR filaments poses new questions regarding the mechanism of XOR in the cell, whether this is a resting state or a hyperactive of the enzyme or whether the filaments are favoured in the presence of substrate or product. This study also demonstrated how an efficient expression system might be used to study clinically relevant mutations as well as conduct inhibition studies on the human enzyme. The structure obtained from X-ray crystallography and cryoEM data may be employed for the design of novel inhibitors that target the human enzyme.