Purine Hydroxylase Research Articles

Regulation of purine hydroxylase and xanthine dehydrogenase from Clostridium purinolyticum in response to purines, selenium, and molybdenum.

The discovery that two distinct enzyme catalysts, purine hydroxylase (PH) and xanthine dehydrogenase (XDH), are required for the overall conversion of hypoxanthine to uric acid by Clostridium purinolyticum was unexpected. In this reaction sequence, hypoxanthine is hydroxylated to xanthine by PH and then xanthine is hydroxylated to uric acid by XDH. PH and XDH, which contain a labile selenium cofactor in addition to a molybdenum cofactor, flavin adenine dinucleotide, and FeS centers, were purified and partially characterized as reported previously. In the present study, the activities of these two enzymes were measured in cells grown in media containing various concentrations of selenite, molybdate, and various purine substrates. The levels of PH protein in extracts were determined by immunoblot assay. The amount of PH protein, as well as the specific activities of PH and XDH, increased when either selenite or molybdate was added to the culture medium. PH levels were highest in the cells cultured in the presence of either adenine or purine. XDH activity increased dramatically in cells grown with either xanthine or uric acid. The apparent increases in protein levels and activities of PH and XDH in response to selenium, molybdenum, and purine substrates demonstrate that these enzymes are tightly regulated in response to these nutrients.

Selenium-dependent metabolism of purines: A selenium-dependent purine hydroxylase and xanthine dehydrogenase were purified from Clostridium purinolyticum and characterized.

During purification of the selenium-dependent xanthine dehydrogenase (XDH) from Clostridium purinolyticum, another hydroxylase was uncovered that also contained selenium and exhibited similar spectral properties. This enzyme was purified to homogeneity. It uses purine, 2OH-purine, and hypoxanthine as substrates, and based on its substrate specificity, this selenoenzyme is termed purine hydroxylase (PH). The product of hydroxylation of purine by PH is xanthine. A concomitant release of selenium from the enzyme and loss of catalytic activity on treatment with cyanide indicates that selenium is essential for PH activity. Selenium-dependent XDH, also purified from C. purinolyticum, was found to be insensitive to oxygen during purification and to use both potassium ferricyanide and 2,6-dichloroindophenol as electron acceptors. Selenium is required for the xanthine-dependent reduction of 2, 6-dichloroindophenol by XDH. Kinetic analyses of both enzymes revealed that xanthine is the preferred substrate for XDH and purine and hypoxanthine are preferred by PH. This characterization of these selenium-requiring hydroxylases involved in the interconversion of purines describes an extension of the pathway for purine fermentation in the purinolytic clostridia.

Cloning and Molecular Characterization of hxA, the Gene Coding for the Xanthine Dehydrogenase (Purine Hydroxylase I) of Aspergillus nidulans

We have cloned and sequenced the hxA gene coding for the xanthine dehydrogenase (purine hydroxylase I) of Aspergillus nidulans. The gene codes for a polypeptide of 1363 amino acids. The sequencing of a nonsense mutation, hxA5, proves formally that the clones isolated correspond to the hxA gene. The gene sequence is interrupted by three introns. Similarity searches reveal two iron-sulfur centers and a NAD/FAD-binding domain and have enabled a consensus sequence to be determined for the molybdenum cofactor-binding domain. The A. nidulans sequence is a useful outclass for the other known sequences, which are all from metazoans. In particular, it gives added significance to the missense mutations sequenced in Drosophila melanogaster and leads to the conclusion that while one of the recently sequenced human genes codes for a xanthine dehydrogenase, the other one must code for a different molybdenum-containing hydroxylase, possibly an aldehyde oxidase. The transcription of the hxA gene is induced by the uric acid analogue 2-thiouric acid and repressed by ammonium. Induction necessitates the product of the uaY regulatory gene.

Characterization of Nitrate Reductase Deficient Mutants of Chlorella sorokiniana

After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO(3) (-) as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH(4) (+)-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein.

Characterization of purine hydroxylase I from Aspergillus nidulans.

Purine hydroxylase I from Aspergillus nidulans was purified 850-fold. The purified preparations exhibited the spectral and catalytic properties, including broad specificity for oxidizing and reducing substrates, typical of molybdenum/flavin/iron-sulphur-containing hydroxylases (oxotransferases).

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 ( g av = 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 (Fe 2S 2)I with g av < 2.0 and the other (Fe 2S 2)II with g av>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.

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 p I 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 K m for hypoxanthine was 119 μ m, for nicotinic acid was 136 μ m, and for NAD + was 525 μ m.

A possible regulatory gene for the molybdenum-containing cofactor in Aspergillus nidulans.

Aspergillus nidulans has three molybdoenzymes, nitrate reductase, purine hydroxylase I and purine hydroxylase II. These three enzymes share a molybdenum-containing cofactor whose synthesis requires the integrity of five loci, designated cnxABC, cnxE, cnxF, cnxG and cnxH. Here we report the existence of a sixth locus, designated cnxJ, which might be involved inthe regulation of cofactor levels. When grown in the presence, but not in the absence, of tungstate or methylammonium, strains carrying cnxJ1 or cnxJ2 have reduced molybdoenzyme levels as judged both from growth properties and enzyme determinations. A new cryosensitive cnxC- allele is also reported. Its phenotype at 37 degrees C (but not 25 degrees C) shows some similarities to that of the two cnxJ- alleles. A structural role for the cnxC (or cnxABC) product in the cofactor is tentatively suggested.

Positive regulation in a eukaryote, a study of the uaY gene of Aspergillus nidulans: I. Characterization of alleles, dominance and complementation studies, and a fine structure map of the uaY--oxpA cluster.

In this paper we characterize genetically a positive eukaryotic regulatory gene: the uaY gene of the ascomycete Aspergillus nidulans. Several steps in the uptake and degradation of purines are under the control of the uaY gene (summarized in Scazzocchio and Gorton 1977). In the present paper 12 uaY-mutations are characterized with respect to their inducibility for adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase and by the absence of the uric acid-xanthine permease scored in vivo by resistance to 2-thiouric acid. While 10 mutations are uniformly unleaky, two others are almost wild type for the induction of urate oxidase. A fine structure map of the uaY gene shows that the two "leaky" mutations are not clustered. The fine structure mapping unambiguously positions six uaY alleles and provides preliminary but interesting trends regarding the pattern of gene conversion in the uaY gene. The enzyme levels in all uaY-/uaY+ heterozygous diploids are intermediate between the corresponding uaY-/uaY- and uaY+/uaY+ homozygous diploids, suggesting that one functional copy of the uaY gene is able to mediate the complete induction of only one set of structural genes. No complementation was found between any two uaY- alleles. This establishes that the mutations showing either of the phenotypes are alleles in the same gene; it fails to provide evidence for intracistronic complementation. A mutation, oxpA5, causes resistance to the xanthine analogue oxypurinol (4,6-dihydroxypyrazolo-(3, 4-d)-pyrimidine) and partial constitutivity of adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase. The constitutive phenotype is suppressed by mutations, blocking the synthesis of intracellular inducers. The mutation is recessive and complements fully with the 11 uaY- mutations tested. It maps to the left of all 12 uaY mutations to which it has been crossed. The data indicate that both the resistance and constitutivity arise from one mutational event in a gene, oxpA, different from uaY and possibly adjacent to it. We propose that the oxpA gene codes for a protein involved in limiting the flow of inducers into the cell nucleus. Thus oxpA and uaY constitute a regulatory gene cluster, indicating that uaY is the regulatory gene.

Product induction of purine hydroxylase II in Aspergillus nidulans

By the use of a mutation, hxB-20, that eliminates purine hydroxylase II activity but retains cross-reacting material and an ancillary NADH dehydrogenase activity, it has been established that 6-hydroxynicotinic acid rather than nicotinic acid, is the true inducer of purine hydroxylase II.

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

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 V 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 ann 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(VI) atom and an--S-group as proposed by Coughlan [FEBS Lett. 81, 1--9 (1977)] 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.

A mutation defective in the xanthine alternative pathway of Aspergillus nidulans

In Aspergillus nidulans uric acid can be produced from xanthine via purine hydroxylase I (xanthine dehydrogenase) or via the xanthine alternative pathway (Darlington and Scazzocchio, Biochem. Biophys. Acta, 166, 569--571; 1968). A mutation defective in the xanthine alternative pathway of Aspergillus nidulans is described. By combining this mutation with hxB-20 which results in complete loss of purine hydroxylase I and II activities, but which conserves cross-reacting material, it is possible to block completely uric acid production and thus investigate which are the effective in vivo inducers of three enzymes under the control of the positive regulatory gene uaY: adenine deaminase, purine hydroxylase I (measured as cross-reacting material) and urate oxidase. It is concluded that uric acid is the only effective physiological inducer, while its 2 and 8 thio-analogues serve as gratuitous inducers.