Purification and Characterization of the FeII- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans

Abstract

His₆-tagged xanthine/α-ketoglutarate (αKG) dioxygenase (XanA) of Aspergillus nidulans was purified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of the two forms of the protein were compared. Evidence was obtained for both N- and O-linked glycosylation on the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacterium-derived XanA is free of glycosylation and behaves as a monomer. Immunological methods identify phosphothreonine in both forms of XanA, with phosphoserine also detected in the bacterium-derived protein. Mass spectrometric analysis confirms glycosylation and phosphorylation of the fungus-derived sample, which also undergoes extensive truncation at its amino terminus. Despite the major differences in the properties of these proteins, their kinetic parameters are similar (k_cat = 30−70 s^-1, K_m of αKG = 31−50 μM, K_m of xanthine ∼ 45 μM, and pH optima at 7.0−7.4). The enzyme exhibits no significant isotope effect when [8-²H]xanthine is used; however, it demonstrates a 2-fold solvent deuterium isotope effect. Cu^II and Zn^II potently inhibit the Fe^II-specific enzyme, whereas Co^II, Mn^II, and Ni^II are weaker inhibitors. NaCl decreases the k_cat and increases the K_m of both αKG and xanthine. The αKG cosubstrate can be substituted with α-ketoadipate (9-fold decrease in k_cat and 5-fold increase in the K_m compared to those of the normal α-keto acid), while the αKG analogue N-oxalylglycine is a competitive inhibitor (K_i = 0.12 μM). No alternative purines effectively substitute for xanthine as a substrate, and only one purine analogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescence of the two enzyme forms by xanthine, αKG, and DHP was used to characterize their binding properties. A XanA homology model was generated on the basis of the structure of the related enzyme TauD (PDB entry 1OS7) and provided insights into the sites of posttranslational modification and substrate binding. These studies represent the first biochemical characterization of purified xanthine/αKG dioxygenase.