Purification and properties of purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a pI of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the Km for hypoxanthine was 119 microM, for nicotinic acid was 136 microM, and for NAD+ was 525 microM.     

Optical and electron paramagnetic resonance spectroscopic studies on purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans contains a molybdenum cofactor very similar to that found in a number of other molybdenum-containing hydroxylases. (A. nidulans contains two purine hydroxylases, I and II, related to each other by possession of a common cofactor and overlapping substrate specificity.) Addition of reducing substrates effects bleaching of the visible absorption spectrum of the enzyme, the decrease in absorbance at 450 nm being linearly proportional to that at 550 nm. No increase in absorption at longer wavelengths was observed during such titrations. Electron paramagnetic resonance studies of reduced samples of native and modified enzyme species showed the presence of a number of Mo(V) signals (gav = 1.97), exhibiting H hyperfine coupling, comparable to those in the corresponding enzymes from other sources. The enzyme possesses two non-heme-iron-sulfur centers, one (Fe2S2)I with gav less than 2.0 and the other (Fe2S2)II with gav greater than 2.0. The flavin radical signal observed at pH 7.8 had a linewidth of 1.5 mT, indicating it to be the anionic form FAD- . In this respect purine hydroxylase II is unique among all molybdenum-containing hydroxylases studied to date.     

The genetic control of the molybdoflavoproteins in Aspergillus nidulans. IV. A comparison between purine hydroxylase I and II.

The purine hydroxylases I and II of Aspergillus nidulans [previously called xanthine dehydrogenases I and II: Scazzocchio, Holl and Foguelman, Eur. J. Biochem. 36, 428--445 (1973)] have been studied in crude extracts. The two enzymes differ in their substrate specificities, purine hydroxylase II being able to accept nicotinate as a substrate and unable to hydroxylate xanthine. The kinetics of inhibition with allopurinol and oxypurinol are also different, the two analogues being pseudo-irreversible inhibitors of purine hydroxylase I, while allopurinol is a competitive inhibitor of purine hydroxylase II and oxypurinol shows anti-competitive inhibition. Differences in electro-phoretic mobility and molecular size are also shown. We have failed to show the formation of hybrid purine hydroxylase I/II molecules. While a common evolutionary origin of the purine hydroxylases could be postulated, the data reveal a considerable divergence.     

A mutant of Asperigillus nidulans lacking NADP-linked glutamate dehydrogenase

Summary A mutation leading to loss of NADP-linked glutamate dehydrogenase pleiotropically leads to derepression of at least some ammonium-repressible activities in Aspergillus nidulans. It confers hypersensitivity to the toxic ammonium analogue methylammonium and maps independently of the two described loci for mutations to methylamonium resistance.     

Functional characterization of a maize purine transporter by expression in Aspergillus nidulans

We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport-deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (K(m) = 30 +/- 2.5 microM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.     

Hydrocortisone induction of phenylalanine hydroxylase isozymes in cultured hepatoma cells.

The hydrocortisone stimulation of phenylalanine hydroxylase activity in Reuber H4 hepatoma cells is shown to be associated with an alteration in phenylalanine hydroxylase isozyme composition. Three forms of phenylalanine hydroxylase were identified in H4 cells which have been treated with hydrocortisone; however, only one of these forms appears to be present prior to glucocorticoid treatment. The relative amounts, as well as the total amount, of the three forms and their chromatographic behavior on hydroxylapatite are nearly identical to the three phenylalanine hydroxylase isozymes found in adult rat liver. The hydroxylase isozyme composition in 2 day old rats is similar to that found in adult rats and in H4 cells treated with hydrocortisone.     

Characterization of active site variants of xanthine hydroxylase from Aspergillus nidulans

Xanthine/α-ketoglutarate (αKG) dioxygenase (XanA) is a non-heme mononuclear Fe^II enzyme that decarboxylates αKG to succinate and CO₂ 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 αKG as measured by fluorescence quenching analysis, in agreement with Lys122 acting to stabilize the binding of αKG. 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 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.