Publications

• Characterization of active site variants of xanthine hydroxylase from Aspergillus nidulans.

  Li M., Mueller T.A., Fraser B.A., Hausinger R.P.

  Xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) is a non-heme mononuclear Fe(II) enzyme that decarboxylates alphaKG to succinate and CO(2) while hydroxylating xanthine to generate uric acid. In the absence of a XanA crystal structure, a homology model was used to target several putative ... >> More

  Xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) is a non-heme mononuclear Fe(II) enzyme that decarboxylates alphaKG to succinate and CO(2) while hydroxylating xanthine to generate uric acid. In the absence of a XanA crystal structure, a homology model was used to target several putative active site residues for mutagenesis. Wild-type XanA and ten enzyme variants were purified from recombinant Escherichia coli cells and characterized. The H149A and D151A variants were inactive and the H340A variant exhibited only 0.17% of the wild-type enzyme activity, consistent with the proposed role of His149, Asp151, and His340 as Fe ligands. The K122A variant led to a 2-fold increase in the K(d) of alphaKG as measured by fluorescence quenching analysis, in agreement with Lys122 acting to stabilize the binding of alphaKG. The N358A variant exhibited a 23-fold decrease in k(cat)/K(m) compared to wild-type XanA, pointing to a key role of Asn358 in catalysis. 9-Methylxanthine was exploited as an alternate substrate, and the C357A, E137A, and D138A variants were found to exhibit relatively enhanced activity consistent with Cys357, Glu137, and Asp138 being proximal to N-9 or involved in its proper positioning. 6,8-Dihydroxypurine was identified as a slow-binding competitive inhibitor of XanA, and significant decreases (E137A and D138A) or increases (Q356A and N358A) in K(i)(app) of the variants were interpreted in terms of distinct interactions between this compound and the corresponding active site side chains. Further support for Cys357 residing at the active site was obtained using thiol-specific reagents that inactivated wild-type enzyme (with partial protection by substrate), whereas the C357A variant was resistant to these reagents. The Q101A, Q356A, and C357A variants showed elevated ferroxidase activity in the absence of substrates, pointing to the presence of the corresponding side chains at the active site. These results confirm most aspects of the homology model and provide additional insight into the enzyme reactivity. << Less

  Arch. Biochem. Biophys. 470:44-53(2008) [PubMed] [EuropePMC]

• Convergent evolution of hydroxylation mechanisms in the fungal kingdom: molybdenum cofactor-independent hydroxylation of xanthine via alpha-ketoglutarate-dependent dioxygenases.

  Cultrone A., Scazzocchio C., Rochet M., Montero-Moran G., Drevet C., Fernandez-Martin R.

  The xanthine oxidases and dehydrogenases are among the most conserved enzymes in all living kingdoms. They contain the molybdopterin cofactor Moco. We show here that in the fungi, in addition to xanthine dehydrogenase, a completely different enzyme is able to catalyse the oxidation of xanthine to ... >> More

  The xanthine oxidases and dehydrogenases are among the most conserved enzymes in all living kingdoms. They contain the molybdopterin cofactor Moco. We show here that in the fungi, in addition to xanthine dehydrogenase, a completely different enzyme is able to catalyse the oxidation of xanthine to uric acid. In Aspergillus nidulans this enzyme is coded by the xanA gene. We have cloned the xanA gene and determined its sequence. A deletion of the gene has the same phenotype as the previously known xanA1 miss-sense mutation. Homologues of xanA exist only in the fungal kingdom. We have inactivated the cognate gene of Schizosaccharomyces pombe and this results in strongly impaired xanthine utilization as a nitrogen source. We have shown that the Neurospora crassa homologue is functionally equivalent to xanA. The enzyme coded by xanA is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase which shares a number of properties with other enzymes of this group. This work shows that only in the fungal kingdom, an alternative mechanism of xanthine oxidation, not involving Moco, has evolved using the dioxygenase scaffold. << Less

  Mol. Microbiol. 57:276-290(2005) [PubMed] [EuropePMC]

• Purification and characterization of the FeII- and alpha-ketoglutarate-dependent xanthine hydroxylase from Aspergillus nidulans.

  Montero-Moran G.M., Li M., Rendon-Huerta E., Jourdan F., Lowe D.J., Stumpff-Kane A.W., Feig M., Scazzocchio C., Hausinger R.P.

  His6-tagged xanthine/alpha-ketoglutarate (alphaKG) 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 glycosyl ... >> More

  His6-tagged xanthine/alpha-ketoglutarate (alphaKG) 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 alphaKG = 31-50 muM, Km of xanthine approximately 45 muM, 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 alphaKG and xanthine. The alphaKG cosubstrate can be substituted with alpha-ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those of the normal alpha-keto acid), while the alphaKG analogue N-oxalylglycine is a competitive inhibitor (Ki = 0.12 muM). 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, alphaKG, 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/alphaKG dioxygenase. << Less

  Biochemistry 46:5293-5304(2007) [PubMed] [EuropePMC]