Purification and Characterization of the FeII- and -Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidu
详细信息 查看全文
- 作者：Gabriela M. Montero-Morá ; n ; Meng Li ; Erika Rendò ; n-Huerta ; Fabrice Jourdan ; David J. Lowe ; Andrew W. Stumpff-Kane ; Michael Feig ; Claudio Scazzocchio ; Robert P. Hausinger
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：May 8, 2007
- 年：2007
- 卷：46
- 期：18
- 页码：5293 - 5304
- 全文大小：323K
- 年卷期：v.46,no.18(May 8, 2007)
- ISSN：1520-4995
文摘
His6-tagged xanthine/
-ketoglutarate (KG) dioxygenase (XanA) of Aspergillus nidulans waspurified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of thetwo forms of the protein were compared. Evidence was obtained for both N- and O-linked glycosylationon the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacterium-derivedXanA 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 theproperties of these proteins, their kinetic parameters are similar (kcat = 30-70 s-1, Km of KG = 31-50 
M, Km of xanthine ~ 45 M, and pH optima at 7.0-7.4). The enzyme exhibits no significant isotopeeffect 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 KG and xanthine. The KG cosubstrate can besubstituted with -ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those ofthe normal -keto acid), while the KG analogue N-oxalylglycine is a competitive inhibitor (Ki =0.12 M). No alternative purines effectively substitute for xanthine as a substrate, and only one purineanalogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescenceof the two enzyme forms by xanthine, KG, 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 (PDBentry 1OS7) and provided insights into the sites of posttranslational modification and substrate binding.These studies represent the first biochemical characterization of purified xanthine/KG dioxygenase.
-ketoglutarate (KG) dioxygenase (XanA) of Aspergillus nidulans waspurified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of thetwo forms of the protein were compared. Evidence was obtained for both N- and O-linked glycosylationon the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacterium-derivedXanA 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 theproperties of these proteins, their kinetic parameters are similar (kcat = 30-70 s-1, Km of KG = 31-50 M, Km of xanthine ~ 45 M, and pH optima at 7.0-7.4). The enzyme exhibits no significant isotopeeffect 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 KG and xanthine. The KG cosubstrate can besubstituted with -ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those ofthe normal -keto acid), while the KG analogue N-oxalylglycine is a competitive inhibitor (Ki =0.12 M). No alternative purines effectively substitute for xanthine as a substrate, and only one purineanalogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescenceof the two enzyme forms by xanthine, KG, 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 (PDBentry 1OS7) and provided insights into the sites of posttranslational modification and substrate binding.These studies represent the first biochemical characterization of purified xanthine/KG dioxygenase.