A Mutation in the Xanthine Dehydrogenase (Purine Hydroxylase I) of Aspergillus nidulans Resulting in Altered Specificity. Implications for the Geometry of the Active Site

Abstract

A point mutation in the structural gene for purine hydroxylase I (xanthine dehydrogenase) of Aspergillus nidulans results in several dramatic pleiotropic effects. The mutant enzyme oxidises 2-hydroxypurine at position 6 rather than 8, shows a 70-fold reduction in the I/ for hypoxanthine, and loses the ability to accept xanthine as a substrate. Allopurinol, a powerful pseudoirreversible inhibitor of the wild type enzyme, behaves as a good substrate of the mutant enzyme. We propose that the substrate and inhibitor specificities of the enzymes depend on the relative position of an orientating site and a catalytic site. All the properties of the mutant enzyme can be explained by assuming that the mutation results in a change of the relative positions of the catalytic and orientating sites.We have assumed that the catalytic site comprises a Mo(V1) atom and an -S-group as proposed by Coughlan [FEBS Lett. 81, 1-9 (1977)l and the orientating site is a lysyl residue. While these assumptions are not strictly necessary for the construction of an abstract geometric model, they are consistent with other data bearing on the structure of the active site of the molybdenum-containing hydroxylases.

Structure and function of the xanthine-oxidase family of molybdenum enzymes

“…The mutation may thus aker the structure considerably, precluding detailed interpretations. Mutants of Aspergillus nidulans pufine hydroxylase are available but require further analysis [93,94].…”

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

“…Mutations mapping in any of the cnx genes, encoding enzymes involved in Moco synthesis (Pateman et al ., 1964; Arst et al ., 1982; Heck et al ., 2002), in hxB , encoding the sulphurase necessary for the cofactor modification (Scazzocchio, 1980; Amrani et al ., 2000) result in loss of both purine hydroxylase I and II activities (Scazzocchio et al ., 1973). The hxA gene mapping in chromosome V is the structural gene for xanthine dehydrogenase (Scazzocchio and Sealy Lewis, 1978; Glatigny and Scazzocchio, 1995), while hxnS , mapping in chromosome VI is the gene encoding purine hydroxylase II (Scazzocchio, 1980; 1994).…”

Characterization of Purine Hydroxylase I from Aspergillus nidulans

“…The biotin-requiring Aspergillus nidulans strain biA-1 used in these studies was kindly provided by Professor Claudio Scazzocchio, Universitk Paris-Sud, Orsay, France. The organism was grown as described previously using 2-thioxanthine (0.5 mg 1-l) to induce synthesis of purine hydroxylase I (Scazzocchio et al, 1973).…”

“…Among the enzymes in this group are the purine hydroxylases I and I1 from Aspergillus nidulans. These have been submitted to thorough genetic analysis Scazzocchio, 1980;Scazzocchio et al, 1973). However, while some properties of purine hydroxylase I have been examined using crude extracts , only purine hydroxylase I1 has been purified and subjected to extensive biochemical characterization and electron paramagnetic resonance spectroscopy .…”