Purification and Characterization of the FeII- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans†Click to copy article linkArticle link copied!
- Gabriela M. Montero-Morán
- Meng Li
- Erika Rendòn-Huerta
- Fabrice Jourdan
- David J. Lowe
- Andrew W. Stumpff-Kane
- Michael Feig
- Claudio Scazzocchio
- Robert P. Hausinger
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 (kcat = 30−70 s-1, Km of αKG = 31−50 μM, Km 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. CuII and ZnII potently inhibit the FeII-specific enzyme, whereas CoII, MnII, and NiII are weaker inhibitors. NaCl decreases the kcat and increases the Km of both αKG and xanthine. The αKG cosubstrate can be substituted with α-ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those of the normal α-keto acid), while the αKG analogue N-oxalylglycine is a competitive inhibitor (Ki = 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.
†
These studies were supported by the National Institutes of Health (Grant GM063584 to R.P.H.), NSF CAREER Grant 0447799 (to M.F.), Université Paris-Sud (including a postdoctoral fellowship to G.M.M.-M.), CNRS and the Institut Universitaire de France (to C.S.), and EU Contract HPRN-CT-1999-084 (XONET to D.J.L. and C.S.).
‡
Université Paris-Sud.
§
These authors contributed equally to this work.
‖
Department of Chemistry, Michigan State University.
⊥
Universidad Nacional Autonoma de Mexico.
@
John Innes Centre.
#
Department of Biochemistry and Molecular Biology, Michigan State University.
+
Institut Universitaire de France.
*
To whom correspondence should be addressed. Telephone: (517) 355-6463, ext. 1610. Fax: (517) 353-8957. E-mail: [email protected].
∞
Department of Microbiology and Molecular Genetics, Michigan State University.
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