Metabolism of pyrimidine deoxyribonucleosides in Neurospora crassa.

The experiments in this report involve the following series of reactions which were previously demonstrated with purified enzyme preparations from Neurospora crassa: thymidine a yields thymine ribonucleoside b yields thymine c yields 5-hydroxymethyluracil d yields 5-formyluracil e yields uracil-5-carboxylic acid f yields uracil. The evidence for some of the reactions occurring in vivo has been incomplete and for others totally lacking. In this paper intact cells of Neurospora are shown to be capable of converting the substrates of each of the reactions to the corresponding products. Studies are described which were carried out in vivo and in vitro with the pyrimidineless strains pyr-4,uc-1,uc-2 and pyr-4,uc-1,uc-3, developed by Williams and Mitchell. The results reported in the present paper indicate that (reaction a) and the uc-3 mutation affects thymine 7-hydroxylase (reactions c,d, and e). Evidence is presented for the 2'-hydroxylase reaction being the major, if not only, way by which Neurospora can initiate the conversion of thymidine to the pyrimidines of nucleic acids and for the 2'-hydroxylation of thymidine and deoxyuridine being catalyzed by the same enzyme. Deoxycytidine was shown not to be hydroxylated in intact cells but instead deaminated to deoxyuridine, which in turn was converted to uridine. Further studies with the uc-3-carrying strain showed that an enzyme other than thymine 7-hydroxylase can also convert 5-formyluracil to uracil-5-carboxylic acid.     

Markedly different ascorbate dependencies of the sequential alpha-ketoglutarate dioxygenase reactions catalyzed by an essentially homogeneous thymine 7-hydroxylase from Rhodotorula glutinis

The alpha-ketoglutarate dioxygenase, thymine 7-hydroxylase (EC 1.14.11.6), has been purified from cultures of Rhodotorula glutinis grown with thymine as a nitrogen source. The purification scheme developed yielded essentially homogeneous preparations of the 7-hydroxylase and also purified another alpha-ketoglutarate dioxygenase, pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3). The purity of the 7-hydroxylase was determined with analytical disc gel electrophoresis in which runs were varied with respect to pH, extent of cross-linking, and the presence of sodium dodecyl sulfate-mercaptoethanol. The 7-hydroxylase apparently exists as a monomer since its molecular weight was 42,700 when determined by molecular gel filtration chromatography and was 40,300 when determined by analytical disc gel electrophoresis under denaturing conditions. Gel filtration chromatography under nondenaturing conditions was used to show that the 2'-hydroxylase has a molecular weight of 64,600. The essentially homogeneous preparations of the 7-hydroxylase were shown to catalyze the thymine-, 5-hydroxymethyluracil-, and 5-formyluracil-dependent oxygenations that are coupled to the decarboxylation of alpha-ketoglutarate, as well as a putative uncoupled decarboxylation which is dependent on uracil. Furthermore, these enzyme preparations were used to show that ATP stimulated the 7-hydroxylase reaction in the absence of ascorbate. Even though it is attractive to consider the four pyrimidine-dependent reactions as being catalyzed by the same active site, they were shown to differ markedly in their dependencies on ascorbate or ATP. The effects of ascorbate and ATP on these reactions, and on the 2'-hydroxylase reaction, are discussed in terms of the possible roles of ascorbate and ATP.     

Genes of the thymidine salvage pathway: thymine-7-hydroxylase from a Rhodotorula glutinis cDNA library and iso-orotate decarboxylase from Neurospora crassa

Genes for two enzymes in the thymidine salvage pathway, thymine-7-hydroxylase (THase; official name thymine dioxygenase) and iso-orotate decarboxylase (IDCase) have been isolated from fungal sources. THase was isolated from a Rhodotorula glutinis cDNA library using a degenerate oligonucleotide based on the published amino acid sequence. The coding sequence was transferred to an Escherichia coli expression system, from which recombinant THase activity was measured using 14C-labeled thymine. The THase sequence shows an almost complete avoidance of codons ending in A or T: 95.8% GC content is present in the third position of codons. A connection between this codon bias and the role of the thymidine salvage pathway in pyrimidine metabolism is proposed. The THase sequence is similar to Group I Fe+2-dependent, alphaKG-dependent dioxygenases. The R. glutinis THase gene was used to locate the probable THase genes in the sequenced genomes of Neurospora crassa and Aspergillus nidulans. The genes neighboring THase in these two genomes are similar to each other, and are similar to the mammalian 2-amino-3-carboxymuconate-6-semialdhyde decarboxylase (ACMSD), leading to their identification as IDCase genes. The N. crassa version was isolated by PCR of genomic DNA, and IDCase activity was measured in recombinant E. coli carrying this gene. A new family of decarboxylases, using similar substrates, is identified by virtue of the protein sequence similarity.     

Thymine 7-hydroxylase and pyrimidine deoxyribonucleoside 2' -hydroxylase activities in Rhodotorula glutinis

Cell-free preparations from Rhodotorula glutinis catalyzed the conversion of deoxyribonucleosides to ribonucleosides in a pyrimidine deoxyribonucleoside 2' -hydroxylase reaction. The reaction occurred with only thymidine or deoxyuridine, of the common deoxyribonucleosides, without detachment of the deoxyribose moiety, at the nucleoside level. The same enzyme preparations catalyzed the conversion of thymine to 5-hydroxymethyluracil in a thymine 7-hydroxylase reaction. Requirements for molecular oxygen, alpha-ketoglutarate, Fe2+, and ascorbate indicated that the 2' -hydroxylase and 7-hydroxylase reactions are of the alpha-keto-acid dioxygenases class. The requirements for alpha-ketoglutarate and Fe2+ were very stringent. During the course of the 2' -hydroxylase and 7-hydroxylase reactions, alpha-ketoglutarate was decarboxylated to form succinate and CO2 so that the ratio of hydroxylated nucleoside or pyrimidine to CO2 was 1:1.5-Hydroxymethyluracil and 5-formyluracil also stimulated the decarboxylation of alpha-ketoglutarate and thus appeared to undergo 7-hydroxylase reactions.     

Separation and characterization of pyrimidine deoxyribonucleoside 2'-hydroxylase and thymine 7-hydroxylase from Aspergillus nidulans

Partially purified preparations from Aspergillus nidulans were shown to catalyze two alpha-ketoglutarate dependent dioxygenase reactions: the pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3) and the thymine 7-hydroxylase (EC 1.14.11.6) reactions. These reactions showed an absolute requirement for alpha-ketoglutarate and molecular oxygen and were stimulated by Fe(II), ascorbate and catalase. Both reactions demonstrated a stoichiometry such that for each mole of substrate (deoxyribonucleoside or pyrimidine) hydroxylated one mole of CO2 was produced from alpha-ketoglutarate. These two activities were separated using DEAE-Sephacel chromatography.     

Uracil's uncoupling of the decarboxylation of alpha-ketoglutarate in the thymine 7-hydroxylase reaction of Neurospora crassa

Highly purified preparations of thymine 7-hydroxylase from Neurospora crassa catalyzed the decarboxylation of alpha-ketoglutarate but yielded no hydroxylated product when uracil was substituted for thymine in the standard incubation mixture. Although the uracil-dependent decarboxylation was much slower than the coupled reaction, both reactions were similar with respect to the requirement for molecular oxygen, the stoichiometric formation of succinate, and the stimulations effected by Fe2+, ascorbate, and catalase. That the same enzyme catalyzed both reactions was indicated by the parallel loss of the uracil- and thymine-dependent activities upon heat denaturation, their copurification, and the lower level of both activities in a mutant strain deficient in the 7-hydroxylase. These data are consonant with molecular oxygen initially attacking alpha-ketoglutarate in the thymine 7-hydroxylase reaction.     

Regulation of pyrimidine salvage in Aspergillus nidulans: a role for the major regulatory gene are A mediating nitrogen metabolite repression

Summary The synthesis of thymine 7-hydroxylase, an -ketoglutarate dependent dioxygenase, is subject both to nitrogen metabolite repression and to oxygen repression, while synthesis of the other pyrimidine salvage pathway dioxygenase, pyrimidine deoxyribonucleoside 2-hydroxylase, is subject to neither. areA300, an allele of the positive acting regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans, considerably elevates levels of thymine 7-hydroxylase, probably alleviating at least partly both nitrogen metabolite repression and oxygen repression. areA300 has little or no effect on levels of pyrimidine deoxyribonucleoside 2-hydroxylase but does elevate net uptake capacities for thymine, thymidine and deoxyuridine two-fold. areA300 was selected as allowing thymine to supplement a pyrimidine auxotrophy and was found to allow supplementation by thymidine, other pyrimidine nucleosides and pyrimidine salvage intermediates as well. This is the first reported evidence for areA control over an activity(-ies) not directly concerned with nitrogen source utilization.     

Mutants affecting thymidine metabolism in Neurospora crassa

When (14)C-thymidine labeled only in the ring is administered to Neurospora crassa, the majority of the recovered label is found in the ribonucleic acid (RNA). Three mutants were isolated in which different steps are blocked in the pathway that converts the pyrimidine ring of thymidine to an RNA precursor. Evidence from genetic, nutritional, and accumulation studies with the three mutants shows the pathway to proceed as follows: thymidine --> thymine --> 5-hydroxymethyluracil --> 5-formyluracil --> uracil --> uridylic acid. A mutant strain in which the thymidine to thymine conversion is blocked is unable to metabolize thymidine appreciably by any route, including entry into nucleic acids. This suggests that Neurospora lacks a thymidine phosphorylating enzyme. A second mutation blocks the pathway at the 5-hydroxymethyluracil to 5-formyluracil step, whereas a third prevents utilization of uracil and all compounds preceding it in the pathway. The mutant isolation procedures yielded three other classes of mutations which are proposed to be affecting, respectively, regulation of the thymidine degradative pathway, transport of pyrimidine free bases, and transport of pyrimidine nucleosides.     

Induction and characterization of a microsomal flavonoid 3'-hydroxylase from parsley cell cultures

A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin [(S)-5, 7, 4'-trihydroxyflavanone] to eriodictyol (5, 7, 3', 4'-tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3'-hydroxylase reaction. In contrast prunin (naringenin 7-O-beta-glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyse hydroxylation of 4-coumaric acid to caffeic acid. 3'-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3'-monooxygenase. Pronounced differences in the inhibition of flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, potassium cyanide and N-ethylmaleimide. Irradiation of the cell cultures led to increase of flavonoid 3'-hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light-induction of phenylalanine ammonialyase and cinnamate 4-hydroxylase. In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3'-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH--cytochrome reductase. The results prove that flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase are two different microsomal monooxygenases.     

Catalysis of three sequential dioxygenase reactions by thymine 7-hydroxylase

A purification scheme has been developed for an enzyme, thymine 7-hydroxylase, which appears to catalyze three sequential dioxygenase reactions, i.e., thymine → 5-hydroxymethyluracil → formyluracil → uracil-5-carboxylic acid. The enzyme was purified 1,300-fold from Neurospora crassa and had specific activities of approximately 1200, 600, and 250 U/mg for the respective reactions. Evidence that a single protein catalyzes the three reactions includes: the parallel purification of the three activities throughout the purification scheme, the inhibition of each reaction by the substrates of the other two, the inhibition of the three reactions by uracil, the parallel loss of the three activities upon heat denaturation, and considerations of a mechanism which suggest that a single active site may be involved.     

Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongylus cantonensis (Nematoda: Metastrongyloidea).

Gravid Angiostrongylus cantonensis can utilize radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phosphoribosyltransferase was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridine was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of thymidylate synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de novo synthesis and by utilization of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate synthetase reaction appears to be vital for the growth of the parasite.     

Thymine 7-hydroxylase from Neurospora crassa. Substrate specificity studies.

A partially purified preparation of thymine 7-hydroxylase (thymine, 2-oxoglutarate : oxygen oxidoreductase (7-hydroxylating), EC 1.14.11.6) from Neurospora crassa was incubated with a number of pyrimidines chemically related to tyymine. 1. Pyrimidines with oxygen or sulfur substituents on atoms Nos. 2 and 4 as well as an alkyl group on atom Nos. 1 or 5 were substrates. 2. Km values were determined for 1-methyluracil, 1-ethyluracil, thymine, 6-azathymine, 1-methylthymine, 1-ethylthymine, 5-formyluracil and 5-hydroxymethyluracil. 3. Uracil was identified as one of the metabolites after incubation with 1-methyluracil. The one-carbon metabolite has not been characterized. 4. Several pyrimidines with polar groups on atoms Nos. 2 and 4 were inhibitory. 5. Addition of 1-methyluracil, 1-methylthymine, 1-ethylthymine or 5-hydroxymethyluracil to incubations with thymine and 2-oxo[1-14C1]glutarate did not result in additional formation of 14CO2, indicating that the same enzyme acts on the different compounds. It has previously been found (Bankel, L., Holme, E., Lindstedt, G. and Lindstedt, S. (1972) FEBS Lett. 21, 135-138) that a mutant strain of N. crassa which is devoid of thymine 7-hydroxylase activity also lacks ability to perform the coupled oxygenation of 2-oxoglutarate and 1-methyluracil, 5-hydroxymethyluracil and 5-formyluracil, respectively. It is concluded that one and the same oxygenase is responsible for the activities studied.     

Specificity of nucleoside transport in Neurospora crassa.

The specificity of nucleoside uptake in germinating conidia of Neurospora crassa was investigated by examining the kinetics of [2-14C]uridine and [8-14C]-adenosine uptake in the wild-type, ad-8, and ud-1 pyr-1 strains. The results obtained strongly indicate that nucleoside transport in N. crassa is mediated solely by a general transport system which accepts both purine and pyrimidine nucleosides. Studies directed at characterizing the specificity of the transport system indicate that general structural features of the nucleoside which enhance its efficiency in binding to the transport system include: (i) a purine or pyrimidine as the heterocyclic ring, (ii) an unfunctionalized ribose or 2'-deoxyribose as the sugar unit, (iii) a beta-configuration about the anomeric carbon, (iv) the absence of substituents at C8 in the purine series and at C5 and C6 in the pyrimidine series, (v) the presence of a C5-C6 double bond in the pyrimidine series, and (vi) the absence of a charge on the heterocyclic ring.     

Depression of uracil uptake by ammonium in Neurospora crassa.

The mechanism of uracil uptake and one aspect of its regulation were studied in germinated conidia of Neurospora crassa. Uracil was found to be taken up by a transport mechanism that did not exhibit Michaelis-Menten kinetics. Rather, the kinetic patterns indicated two separate systems or a single transport mechanism with negative cooperativity. Cytosine and thymine inhibited uracil uptake, but uridine did not. The mutant strain uc-5-pyr-1, which failed to transport uracil, was used in reversion studies and to map the uc-5 locus. Spontaneous reversion rates at the uc-5 locus were found to be approximately 2 x 10(-8), indicating that the uc-5 lesion results from a single mutation. Loss of the uracil transport function through a single mutation favors the model of a single transport mechanism with negative cooperativity. Uracil uptake was significantly decreased in the presence of NH 4+, and evidence is presented for repression by NH4+ of a uracil transport system. Growth rates of pyrimidine-requiring and wild-type strains measured in the presence and absence of NH4+, with uracil as the pyrimidine supplement, showed that NH4+ decreased the growth rates of the pyrimidine-requiring strains significantly, while having no effect on wild-type growth rates.     

A closed conformation of Bacillus subtilis oxalate decarboxylase OxdC provides evidence for the true identity of the active site

Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659-7669). The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 3(10) helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Site-directed mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.     

The in silico screening and X-ray structure analysis of the inhibitor complex of Plasmodium falciparum orotidine 5'-monophosphate decarboxylase

Orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) catalyses the final step in the de novo synthesis of uridine 5'-monophosphate (UMP) from orotidine 5'-monophosphate (OMP). A defective PfOMPDC enzyme is lethal to the parasite. Novel in silico screening methods were performed to select 14 inhibitors against PfOMPDC, with a high hit rate of 9%. X-ray structure analysis of PfOMPDC in complex with one of the inhibitors, 4-(2-hydroxy-4-methoxyphenyl)-4-oxobutanoic acid, was carried out to at 2.1 ? resolution. The crystal structure revealed that the inhibitor molecule occupied a part of the active site that overlaps with the phosphate-binding region in the OMP- or UMP-bound complexes. Space occupied by the pyrimidine and ribose rings of OMP or UMP was not occupied by this inhibitor. The carboxyl group of the inhibitor caused a dramatic movement of the L1 and L2 loops that play a role in the recognition of the substrate and product molecules. Combining part of the inhibitor molecule with moieties of the pyrimidine and ribose rings of OMP and UMP represents a suitable avenue for further development of anti-malarial drugs.     

Substitution of nucleoside triphosphates for ascorbate in the thymine 7-hydroxylase reaction of Rhodotorula glutinis

Nucleoside di- and triphosphates substituted for ascorbate in the thymine 7-hydroxylase reaction in studies carried out with purified preparations from Rhodotorula glutinis. The stimulations brought about by ascorbate and ATP were found not to be additive. Studies with analogues of ATP indicated that hydrolysis may not need to occur in order for the nucleotide effect to be expressed. The stoichiometry of the production of 5-hydroxymethyluracil and CO2 was not changed by the substitution of ATP for ascorbate. The 7-hydroxylase was found to have considerable thermal stability, and inactivation at 98 degrees C resulted in a parallel loss of the activities effected by ascorbate and ATP. This and the retention of the nucleotide effect upon purification suggest the effect is not mediated through another protein co-purified with the 7-hydroxylase.     

Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end complex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406-415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.     

Pyrimidine base and ribonucleoside utilization by thePseudomonas alcaligenes group

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of thePseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth ofPseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supportedPseudomonas alcaligenes ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by eitherP. pseudoalcaligenes orP. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts ofP. pseudoalcaligenes andP. alcaligenes. InP. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.