New perspectives on the roles of pyrimidines in the central nervous system.

Since 1956, when exogenous uridine and cytidine were found to be necessary for the maintenance of perfused rat brain function, the co-existence of de novo synthesis, salvage pathways and removal of pyrimidine bases in the CNS has been a controversial subject. Here, we review studies on metabolites and enzymes of pyrimidine metabolism through more than 60 years. In view of known and newly-described inherited pyrimidine and purine disorders - some with complex clinical profiles of neurological impairments - we underline the necessity to investigate how the different pathways work together in the developing brain and then sustain plasticity, regeneration and neuro-transmission in the adult CNS. Experimentally, early incorporation studies in animal brain slices and homogenates with radio-labelled nucleosides or precursors demonstrated salvage activity or de novo synthesis. Later, the nucleoside transporters and organic anionic transporters underlying uptake of metabolites and anti-pyrimidine drugs in the CNS were identified. Recently, the expression of de novo enzymes in glial cells and neurons was verified using (immuno) histochemical and in-situ-hybridization techniques. Adult brain was shown to take up or produce all pyrimidine (deoxy) ribonucleosides or, after uptake and phosphorolysis of nucleosides, to make use of ribose for different purposes, including energy. More recently, non-canonical pyrimidine bases (5mC, 5hmC) have been found most notably in brain, pointing to considerable postreplicative DNA metabolism, with the need for pyrimidine-specific enzymes. Even more perspectives are emerging, with advances in genome analysis and in the manipulation of expression from the gene.

Orotate (orotic acid): An essential and versatile molecule.

Orotate (OA) is well-known as a precursor in biosynthesis of pyrimidines; in mammals it is released from the mitochondrial dihydroorotate dehydrogenase (DHODH) for conversion to UMP by the cytoplasmic UMP synthase enzyme. OA is also a normal part of the diet, being found in milk and dairy products, and it is converted to uridine for use in the pyrimidine salvage pathway predominantly in liver, kidney and erythrocytes. Early research into nutrition identified orotate as "vitamin B13," and its use as a complex with organic cations or metal ions was promulgated in body-building, and in assisting therapies of metabolic syndromes. It has recently been established that the amelioration of gout by dairy products arises from the competition of orotate and urate at the hURAT1 transporter. The orotic aciduria that arises in children with defective UMP synthase can be rescued by oral uridine therapy, since UMP is the end-product and also a feedback inhibitor of the de novo pathway. In contrast, Miller (dysmorphology) syndrome is connected with defects in DHODH, and hence in the supply of OA, and cannot be helped by uridine. Other models of dysmorphisms are connected with enzymes early in the pyrimidine de novo pathway. We conclude that the OA molecule is itself required for the regulation of genes that are important in the development of cells, tissues and organisms.

Reduction of benzaldehyde catalyzed by papain-based semisynthetic enzymes.

Some features of native enzyme's active site were used to conjunction with a chemical reagent or modifying group, which would generate new functionality different from the natural enzyme. In order to obtain an efficient catalyst, we have designed four different molecular size N-derivatives of modifiers and introduced them into the active site of papain to obtain new semisynthetic enzymes, which were used as catalyst in reduction of benzaldehyde to yield benzyl alcohol respectively, and the reactions carried out with recycling agent in 0.1 M phosphate buffer pH 6.5 at 37 degrees C. The results had shown that a longer N-derivative of semisynthetic enzyme had higher catalytic activity. Furthermore, we propose a plausible model for the catalytic mechanism in the semisynthetic enzymes system.

4-pyridone-3-carboxamide ribonucleoside triphosphate accumulating in erythrocytes in end stage renal failure originates from tryptophan metabolism.

We recently identified an erythrocyte nucleotide accumulating in end-stage renal disease as 4-pyridone-3-carboxamide ribonucleotide triphosphate (4PYTP), a nucleotide never described previously. Plasma tryptophan concentration has been previously reported to be reduced in patients in chronic renal failure that is in turn associated with elevated precursors of tryptophan metabolism, including L -kynurenine and quinolinic acid, both of which have been implicated in the neurotoxic manifestations of chronic renal failure. Here we compare mean erythrocyte 4PYTP, and plasma tryptophan concentrations, in controls and four patient groups with renal impairment (10 per group) and confirmed a reduction in plasma tryptophan in patients on dialysis that corrected with renal transplantation. We found: An inverse correlation between plasma tryptophan and red cell 4PYTP concentrations (R(2)=0.44, P<0.001) when all patients were grouped together. Restoration of both tryptophan and 4PYTP concentrations to control values was only achieved following renal transplantation. 4PYTP was absent from erythrocytes in Molybdenum cofactor (MoCF) deficiency implicating aldehyde oxidase/dehydrogenase, a Molybdenum requiring enzyme. High 4PYTP erythrocyte concentrations in adenine or hypoxanthine-phosphoribosyltransferase deficient patients in severe uremia (113 microM and 103 microM), confirmed the lack of involvement of either enzyme in 4PYTP formation. We propose that 4PYTP is formed by a novel route involving the oxidation of the intermediates of NAD turnover from quinolinic acid by aldehyde oxidase.

Purine and pyrimidine metabolism: a firm basis for a transformed society.

Purines and pyrimidines form the backbone of DNA and RNA. Hence, modification of purine and pyrimidine metabolism can have serious effects on normal functioning of a subject. These aspects formed the main topics for an International and a European Series of meetings, dedicated to the metabolism in man. In order to streamline the organization of these meetings the European Society was transformed to an International society: the Purine and Pyrimidine Society (www.ppsociety.org). This special issue of Nucleosides, Nucleotides, and, Nucleic, Acids highlights the last European meeting in Prague, focusing on inborn errors, cardiac diseases, inflammatory diseases, rheumatology, haematology, cancer, virology, genetic polymorphism, specific methodology, and, of course, metabolism. The meeting in Chicago in 2007 will be the first meeting of the Purine and Pyrimidine Society.

The novel nucleotide 4KNTP, in high concentrations in erythrocytes of renal failure children: a comparison with accumulation of other putative precursors in the plasma.

We have measured the concentrations of metabolites related to the turnover of NAD, which accumulate in the blood of children with renal failure. One is a novel nucleotide, identified as the N1-riboside triphosphate of 4-pyridone-3-carboxamide (4PYTP), also described as 4KNTP, which accumulates in the erythrocytes in parallel with renal failure.

An unusual pyridine nucleotide accumulating in erythrocytes: its identity and positive correlation with degree of renal failure.

We have investigated an unusual nucleotide that accumulates, with precursors, in the erythrocytes of patients in uraemia. This nucleotide is related chemically to the NAD breakdown product, N1-methyl-2-pyridone-5-carboxamide (Me2Py), found in high concentrations in the plasma of uraemic patients. Both Me2Py and the nucleotide accumulate to high concentrations in the blood during uraemia: our investigations of samples from renal out-patients have provided information on a plausible link between the two compounds.

GTP concentrations are elevated in erythrocytes of renal transplant recipients when conventional immunosuppression is replaced by the inosine monophosphate dehydrogenase inhibitor mycophenolic acid mofetil (MMF).

We show that GTP concentrations rise in the erythrocytes of renal transplant recipients receiving the immunosuppressant MMF, and demonstrate that this effect is not caused by poor renal function after engraftment. We propose a model that is consistent with our observations.

Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa.

Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.

Simultaneous separation by high-performance liquid chromatography of carbamoyl aspartate, carbamoyl phosphate and dihydroorotic acid.

Leflunomide is an immunomodulatory drug which acts by inhibiting dihydroorotic acid dehydrogenase, the fourth enzyme of pyrimidine biosynthesis. We modified our high-performance liquid chromatography method to demonstrate that the principal metabolite in mitogen-stimulated human T-lymphocytes incubated with leflunomide was not dihydroorotic acid, but carbamoyl aspartate. Identification involved preparation of [14C]carbamoyl aspartate from [14C]aspartic acid and mammalian aspartate transcarbamoylase. Accumulation of carbamoyl aspartate indicates that under these conditions the equilibrium constant for dihydroorotase favours the reverse reaction. This HPLC method, enabling simultaneous separation of the first four intermediates in the de novo pyrimidine pathway may be of use in a variety of experimental situations.

Leflunomide inhibits pyrimidine de novo synthesis in mitogen-stimulated T-lymphocytes from healthy humans.

The mode of action of Leflunomide, an immunomodulatory drug used in rheumatoid arthritis, is debated. This study, using 14C-labeled de novo purine and pyrimidine synthesis precursors, proves conclusively that the prime target in proliferating human T-lymphocytes is pyrimidine biosynthesis at the level of dihydroorotic-acid dehydrogenase. Leflunomide (25 and 50 microM), like Brequinar (0.5 and 1 microM), a demonstrated dihydroorotic-acid dehydrogenase inhibitor, was cytostatic, not cytotoxic, with proliferation being halted in the G1 phase. Both drugs restricted the normal 4-8-fold mitogen-induced expansion of pyrimidine pools over 72 h to concentrations found in nonstimulated T-cells and [14C]bicarbonate incorporation into UTP, ATP, and GTP. Uridine (50 microM) restored expansion of all pools, but [14C]bicarbonate incorporation into ATP and GTP only, not UTP. [14C]Hypoxanthine salvage was also restricted, indicating that purine salvage pathways are compromised likewise by both inhibitors. [14C]Glycine studies confirmed that restriction of de novo purine synthesis occurred secondary to inhibition of proliferation since this was reversed by uridine rescue, except at 100 microM Leflunomide. 100 microM Leflunomide markedly depleted ATP and GTP pools also, which would have serious consequences for ATP-dependent enzymes essential to the immune response, thereby explaining non-pyrimidine-related effects reported for Leflunomide at 100 microM and above.

A reciprocal allosteric mechanism for efficient transfer of labile intermediates between active sites in CAD, the mammalian pyrimidine-biosynthetic multienzyme polypeptide.

Carbamoyl phosphate is the product of carbamoyl phosphate synthetase (CPS II) activity and the substrate of the aspartate transcarbamoylase (ATCase) activity, each of which is found in CAD, a large 240-kDa multienzyme polypeptide in mammals that catalyses the first three steps in pyrimidine biosynthesis. In our study of the transfer of the labile intermediate between the two active sites, we have used assays that differentiate the synthesis of carbamoyl phosphate from the overall reaction of CPS II and ATCase that produces carbamoyl aspartate. We provided excess exogenous carbamoyl phosphate and monitored its access to the respective active sites through the production of carbamoyl phosphate and carbamoyl aspartate from radiolabelled bicarbonate. Three features indicate interactions between the folded CPS II and ATCase domains causing reciprocal conformational changes. First, even in the presence of approximately 1 mM unlabelled carbamoyl phosphate, when the aspartate concentration is high ATCase uses endogenous carbamoyl phosphate for the synthesis of radiolabelled carbamoyl aspartate. In contrast, the isolated CPS II forward reaction is inhibited by excess unlabelled carbamoyl phosphate. Secondly, the affinity of the ATCase for carbamoyl phosphate and aspartate is modulated when substrates bind to CPS II. Thirdly, the transition-state analogue phosphonacetyl-L-aspartate is a less efficient inhibitor of the ATCase when the substrates for CPS II are present. All these effects operate when CPS II is in the more active P state, which is induced by high concentrations of ATP and magnesium ions and when 5'-phosphoribosyl diphosphate (the allosteric activator) is present with low concentrations of ATP; these are conditions that would be met during active biosynthesis in the cell. We propose a phenomenon of reciprocal allostery that encourages the efficient transfer of the labile intermediate within the multienzyme polypeptide CAD. In this model, binding of aspartate to the active site of ATCase causes a conformational change at the active site of the liganded form of CPS II, which protects it from inhibition by its product, carbamoyl phosphate; reciprocally, the substrates for CPS II affect the active site of ATCase by increasing the affinity for its substrates, endogenous carbamoyl phosphate and aspartate, and thus impede access of exogenous carbamoyl phosphate or the transition-state analogue. Reciprocal allostery justifies the close association of the enzyme activities within the polypeptide and ensures that carbamoyl phosphate is efficiently synthesised and is dedicated to the second step of pyrimidine biosynthesis. These conditions fulfill those required for metabolic channeling in the cell.

Mammalian dihydroorotase; secondary structure, and interactions with other proteolytic fragments from the multienzyme polypeptide CAD.

We have purified mammalian dihydroorotase as a polypeptide fragment of 46 kDa from an elastase digest of CAD, the 240-kDa multienzyme that catalyses the first three reactions of pyrimidine biosynthesis. The thermal unfolding of the domain was analysed through the change in circular dichroism, indicating a sharp transition at 45 degrees C in which most of the native alpha-helix is lost. Although there is good evidence that the fragments associate as dimers in solution, chemical cross-linking was only possible when the dihydroorotase domain was included in a larger proteolytic fragment of 190-195 kDa. Cross-linking of the isolated domain yielded a species that appeared to result from links between two or more sub-domains, and did not yield the expected 90-kDa dimer of dihydroorotase. We speculate that the presence of other folded regions of CAD stabilises the interactions between dihydroorotase domains.

Proteolytic cleavage of the multienzyme polypeptide CAD to release the mammalian aspartate transcarbamoylase. Biochemical comparison with the homologous Escherichia coli catalytic subunit.

We have demonstrated biochemically that the conformation of the proteolytic fragment (mammalian aspartate transcarbamoylase) from the C-terminus of the 240-kDa multienzyme polypeptide carrying the activities carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydroorotase (CAD) is similar to that of the catalytic subunits from Escherichia coli aspartate transcarbamoylase. We have measured the extent of unfolding of the mammalian aspartate transcarbamoylase in guanidinium chloride solutions, and have also demonstrated that the protein cross-reacts with antibodies raised against the E. coli enzyme. CAD is digested by low concentrations of trypsin in the presence of 0.2 mM UTP to release an active aspartate transcarbamoylase domain and a 195-kDa 'nicked CAD' molecule containing active carbamoyl phosphate synthetase. These two products are easily separated by ion-exchange chromatography. Similar proteolytic cleavage and trimming by elastase releases a family of aspartate transcarbamoylase fragments. Direct N-terminal sequencing of the aspartate transcarbamoylase fragments confirms predictions of the most accessible residues in the region linking the aspartate transcarbamoylase and dihydroorotase domains. Only the largest of the four fragments generated by elastase retains phosphorylation site 2. When this largest fragment is phosphorylated, the family of aspartate transcarbamoylase fragments is eluted together from ion-exchange columns in a different fraction from the completely unphosphorylated preparation, demonstrating the affinity of the domains for each other.

A protonated histidine residue in a phosphorylation site for cyclic AMP-dependent protein kinase. Comparison of a synthetic peptide with the exposed linking region in the multienzyme polypeptide CAD.

The multienzyme polypeptide CAD is phosphorylated at two sites by cyclic AMP (cAMP)-dependent protein kinase. Site 2 has two interesting features: it is located in a 'linking region' between two discretely folded enzyme domains, and a histidine, instead of the more usual arginine, is found three positions N-terminal to the phosphorylated serine. A synthetic peptide corresponding to the sequence around site 2 has an extended or random structure in solution, and the proton n.m.r. chemical shift of the histidine residues can be titrated against pH in the range 6.0-8.0. The peptide is phosphorylated more rapidly by cAMP-dependent protein kinase at lower pH values, indicating that the protonated histidine side chain corresponds to the arginine in the consensus recognition sequence for the kinase. Kemptide, a specific synthetic substrate for the kinase, was phosphorylated with a higher affinity and at a similar rate at all pH values. CAD was a better substrate than the synthetic peptide, and labelling was not affected by the pH of the incubation conditions. The results indicate that the phosphorylation site in the interdomain linker is sufficiently exposed to the solvent to ensure accessibility to the kinase, but that secondary or tertiary structure in the intact protein allows the histidine residue to remain protonated at physiological pH and enhances recognition of the phosphorylatable serine residue.