Convergent evolution of hydroxylation mechanisms in the fungal kingdom: molybdenum cofactor‐independent hydroxylation of xanthine via α‐ketoglutarate‐dependent dioxygenases

Abstract

SummaryThe 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 a a a a -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.

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

“…Three loops in the sequence, comprising residues 72-88, 173-190, and 219-231, had no counterparts in TauD and were not modeled (indicated by boxes at the appropriate positions in the figure), but these are all distant from the putative active site region. The homology model contains the Fe II -binding site (His149, Asp151, and His340) expected from prior sequence alignments (2). The cosubstrate (shown in yellow) was positioned into the model so as to chelate Fe II in a similar fashion as αKG occurs in TauD.…”

“…In 2005, a novel mechanism for xanthine metabolism was discovered in certain fungi (2). This finding arose out of the observation that all mutants of Aspergillus nidulans defective in xanthine dehydrogenase (i.e., with mutations affecting the structural gene hxA, the cnx genes for Moco synthesis, or hxB for sulfuration of Moco) retained the ability to grow on xanthine as sole nitrogen source (2,3).…”

“…This homology suggested that the alternative xanthine oxidation mechanism present in some fungi might utilize an Fe IIdependent xanthine/αKG dioxygenase. Such activity was demonstrated in both crude and partially purified extracts of fungal mycelia of strains that expressed the xanA gene (2). On the basis of the previously described mechanism for Fe II /αKG hydroxylases (6), XanA is proposed to catalyze the series of reactions (αKG binding, xanthine binding, O 2 binding, αKG decomposition and O-O cleavage to yield an Fe IV -oxo species, hydrogen atom abstraction, and hydroxyl radical rebound) depicted in Scheme 1.…”

“…This finding arose out of the observation that all mutants of Aspergillus nidulans defective in xanthine dehydrogenase (i.e., with mutations affecting the structural gene hxA, the cnx genes for Moco synthesis, or hxB for sulfuration of Moco) retained the ability to grow on xanthine as sole nitrogen source (2,3). A mutation affecting this alternative process was identified, and the cognate gene xanA was localized to chromosome VIII (4).…”

“…In the fungal kingdom, this enzyme coexists with the classical xanthine hydroxylase; i.e., some fungi possess both xanthine hydroxylase and xanthine/αKG dioxygenase, while others possess only one or the other. Notably, yeasts as evolutionarily distant as S. pombe and Kluveromyces lactis are able to metabolize xanthine through the activity of a XanA homologue (2). They lack a classical xanthine dehydrogenase, and they are incapable of synthesizing Moco, which is universally present in the classical xanthine hydroxylases.…”