Functional analysis of the pyrimidine de novo synthesis pathway in solanaceous species

Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.     

In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. I. Catalytic and regulatory properties of aspartate transcarbamylase

A permeabilization procedure was adapted to allow the in situ determination of aspartate transcarbamylase activity in Saccharomyces cerevisiae. Permeabilization is obtained by treating cell suspensions with small amounts of 10% toluene in absolute ethanol. After washing, the cells can be used directly in the enzyme assays. Kinetic studies of aspartate transcarbamylase (EC 2.1.3.2) in such permeabilized cells showed that apparent Km for substrates and Ki for the feedback inhibitor UTP were only slightly different from those reported using partially purified enzyme. The aspartate saturation curve is hyperbolic both in the presence and absence of UTP. The inhibition by this nucleotide is noncompetitive with respect to aspartate, decreasing both the affinity for this substrate and the maximal velocity of the reaction. The saturation curves for both substrates give parallel double reciprocal plots. The inhibition by the products is linear noncompetitive. Succinate, an aspartate analog, provokes competitive and uncompetitive inhibitions toward aspartate and carbamyl phosphate, respectively. The inhibition by phosphonacetate, a carbamyl phosphate analog, is uncompetitive and noncompetitive toward carbamyl phosphate and aspartate, respectively, but pyrophosphate inhibition is competitive toward carbamyl phosphate and noncompetitive toward aspartate. These results, as well as the effect of the transition state analog N-phosphonacetyl-L-aspartate, all exclude a random mechanism for aspartate transcarbamylase. Most of the data suggest an ordered mechanism except the substrates saturation curves, which are indicative of a ping-pong mechanism. Such a discrepancy might be related to some channeling of carbamyl phosphate between carbamyl phosphate synthetase and aspartate transcarbamylase catalytic sites.     

Analysis of lines of mice selected for fat content. 3. Flux through the de novo lipid synthesis pathway

The flux through the de novo fatty acid synthesis pathway was estimated in lines of mice which differed substantially in fat content following 26 generations of selection at 10 weeks of age. Previous estimates of lipogenic enzyme activities had indicated an increase in the capacity for lipogenesis in the Fat compared to the Lean line. Therefore the in vivo flux in lipogenesis was measured in both liver and gonadal fat pad (GFP) tissues of males at 5 and 10 weeks of age, using the rate of incorporation of 3H from 3H2O and 14C from acetate and citrate into total lipids. At both ages and in both tissues the Fat line had a higher flux, about 20% increase in the liver and up to three-fold increase (range 1.2- to 3.4-fold) in the GFP. We conclude that direct selection for fatness in mice has resulted in metabolic changes in the rate of de novo fatty acid synthesis, and that the changes are largely detectable before 10 weeks, the age of selection.     

Analysis of pyrimidine synthesis de novo intermediates in urine during crisis of a patient with ornithine transcarbamylase deficiency

Analysis of pyrimidine synthesis de novo intermediates and pyrimidine degradation products in urine samples from a decompensated patient with an ornithine transcarbamylase deficiency showed a strikingly aberrant metabolic profile. Strongly elevated levels of N-carbamyl-aspartate, orotate and uracil were present whereas the concentration of uridine was only marginally increased. The level of pyrimidine excretion appeared to be independent of the ammonia levels in blood, which were only mildly increased.     

Comparison of van 't Hoff and calorimetrically determined enthalpies of binding of N-phosphonacetyl-L-aspartate to E. coli aspartate transcarbamylase.

A comparison has been made of the values obtained by direct calorimetric measurements and van 't Hoff analysis, under similar conditions, for the enthalpy of binding of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to E. coli aspartate transcarbamylase and its catalytic subunit. In the case of the catalytic subunit, data were obtained at both saturating and non-saturating concentrations of L-Asp, and at two ionic strengths. Despite a 1000-fold difference in protein concentrations, and the obligatory omission of carbamyl phosphate in the calorimetric experiments, the values obtained by the two methods are shown to agree to within 15% when appropriate corrections are made. These results suggest that subunit dissociation is not a significant factor at the low protein concentrations used in the van 't Hoff analysis, and, conversely, that aggregation of the protein is negligible at the high protein concentrations used in the calorimetric experiments. They also imply that, at pH 8.3, the enthalpic difference between the two conformational states of the enzyme which exist in the presence and absence of substrates is less than 2.5 kcal/mol. In addition, the trends in the three sets of data for the catalytic subunit indicate that ionic bonds are involved in binding PALA to the active site, and that non-productive binding by L-Asp is negligible under these experimental conditions.     

Subcellular localization of the pathway of de novo pyrimidine nucleotide biosynthesis in pea leaves

The subcellular distribution of the enzymes of de novo pyrimidine nucleotide biosynthesis was investigated in pea (Pisum sativum L. cv Progress No. 9) leaves. Aspartate carbamoyltransferase, the committed step of the pathway, was found to be strictly confined to the chloroplasts. Dihydro-orotase, orotate phosphoribosyl transferase, and orotidine decarboxylase activities were also found only in the plastids. The remaining enzyme of the pathway, dihydroorotate dehydrogenase, was shown to be mitochondrial.     

An anomaly of the genetic regulation of the de novo pyrimidine pathway in the plant Arabidopsis

Humans afflicted by hereditary orotic aciduria are characterized by insufficiencies in the de novo pyrimidine pathway. Mutants at a nuclear gene locus in Arabidopsis, in contrast, exhibit increased activities of orotidylic acid (O5P) pyrophosphorylase (2.4.2.10) and O5P-decarboxylase (4.1.1.23). In the plants, as well as in human cells, the symptoms of the genetic disorder can be partly cured by feeding the pyrimidine analogue 6-azauracil. In normal human cells, the supply of the antimetabolite 6-azauridine leads to augmented levels of these enzymes, and in the cell cultures of patients suffering from orotic aciduria type I nearly normal levels of the enzymes are observed. In the tissues of Arabidopsis, on 6-azauracil administration, the level of O5P-pyrophosphorylase decreases while that of O5P-decarboxylase is elevated. The genetic alteration may involve a regulatory function in both humans and plants.Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 6802. Approved.     

Superproduction and rapid purification of Escherichia coli aspartate transcarbamylase and its catalytic subunit under extreme derepression of the pyrimidine pathway

A strain of Escherichia coli has been constructed which greatly overproduces the enzyme aspartate transcarbamylase. This strain has a deletion in the pyrB region of the chromosome and also carries a leaky mutation in pyrF. Although this strain is a pyrimidine auxotroph, it will grow very slowly without pyrimidines if a plasmid containing the pyrB gene is introduced into it. Derepression occurs when this strain exhausts its uracil supply during exponential growth. Under extreme derepression, aspartate transcarbamylase can account for as much as 60% of the total cellular protein. This host strain/plasmid system can be utilized for the rapid purification of wild-type aspartate transcarbamylase or plasmid-born mutant versions of the enzyme. This system is particularly well-suited for analysis of the latter since the control of overproduction resides exclusively on the bacterial chromosome. Therefore, any plasmid bearing the pyrBI operon can be made to overproduce aspartate transcarbamylase in this host strain. Based on this system, a rapid purification procedure has been developed for E. coli aspartate transcarbamylase. The purification scheme involves an ammonium sulfate fractionation followed by a single precipitation of the enzyme at its isoelectric point. In a similar fashion, this strain can also be employed to produce exclusively the catalytic subunit of the enzyme if the plasmid only carries the pyrB gene. This system may be adapted to overproduce other proteins as well by using this host strain and the strong pyrB promoter linked to another gene.     

In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. 2. Reaction mechanism of aspartate transcarbamylase dissociated from carbamylphosphate synthetase by genetic alteration

The reaction mechanism of Saccharomyces cerevisiae aspartate transcarbamylase was studied in permeabilized cells of a mutant in which this enzyme is not associated to carbamylphosphate synthetase. The results obtained indicate an ordered mechanism in which carbamylphosphate binds first, followed by aspartate, with dissociation of the products in the order phosphate then carbamylaspartate. Interestingly, this clear-cut mechanism differs from the more complex behavior shown by aspartate transcarbamylase when this enzyme is associated to carbamylphosphate synthetase in wild-type S. cerevisiae (B. Penverne and G. Hervé, Arch. Biochem. Biophys. (1983) 225, 562-575). This difference indicates that the association of the two enzymes within the multienzymatic complex alters the apparent kinetic properties of aspartate transcarbamylase. Such an enzyme-enzyme interaction might be related to the channeling of carbamylphosphate from one catalytic site to the other one.     

A new folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis

5,10-Dideazatetrahydrofolate (DDATHF) is a new antimetabolite designed as an inhibitor of folate metabolism at sites other than dihydrofolate reductase. DDATHF was found to inhibit the growth of L1210 and CCRF-CEM cells in culture at concentrations in the range of 10-30 nM. The inhibitory effect of DDATHF on the growth of L1210 and CCRF-CEM cells was reversed by either hypoxanthine or aminoimidazole carboxamide. Growth inhibition by DDATHF was prevented by addition of both thymidine and hypoxanthine, but not by thymidine alone. 5-Formyltetrahydrofolate reversed the effects of DDATHF in a dose-dependent manner. DDATHF had no appreciable inhibitory activity against either dihydrofolate reductase or thymidylate synthase in vitro, but was found to be an excellent substrate for folylpolyglutamate synthetase. DDATHF had little or no effect on incorporation of either deoxyuridine or thymidine into DNA, in distinct contrast to the effects of the classical dihydrofolate reductase inhibitor, methotrexate. DDATHF was found to deplete cellular ATP and GTP over the same concentrations as those inhibitory to leukemic cell growth, suggesting that the locus of DDATHF action was in the de novo purine biosynthesis pathway. The synthesis of formylglycinamide ribonucleotide in intact L1210 cells was inhibited by DDATHF with the same concentration dependence as inhibition of growth. This suggested that DDATHF inhibited glycinamide ribonucleotide transformylase, the first folate-dependent enzyme of de novo purine synthesis. DDATHF is a potent folate analog which suppresses purine synthesis through direct or indirect inhibition of glycinamide ribonucleotide transformylase.     

Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.     

Enzymes of de novo pyrimidine biosynthesis in Babesia rodhaini

The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2); dihydroorotase (DHOase: E.C. 3.5.2.3); dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5'-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO-DHase, OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacetyl)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent Ki of 7 microM. Dihydro-5-azaorotate inhibited the DHO-DHase (Ki, 16 microM) and 5-azaorotate (Ki, 21 microM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (Ki, 0.3 microM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product, UMP (Ki, 120 microM) and the purine nucleotide, XMP (Ki, 95 microM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.     

Pyridoxal 5'-phosphate, a fluorescent probe in the active site of aspartate transcarbamylase.

Pyridoxal-P reacts specifically with a single lysine residue at the active site of Escherichia coli aspartate transcarbamylase (Greenwell, P., Jewett, S. L., and Stark, G. R. (1973) J. Biol. Chem. 248, 5994-6001). Reduction of the Schiff base with sodium borohydride, succinylation of the remaining lysine residues, and digestion with trypsin result in formation of a single pyridoxyl peptide, which was purified to homogeneity after chromatography on DEAE-cellulose, treatment with alkaline phosphatase, and rechromatography. Amino acid composition and the results of limited sequential degradation showed that this peptide corresponds to residues 62 to 98 in the sequence of Konigsberg and co-workers, and contains 2 residues of lysine (Henderson, L., Roy, D., Martin, D., and Konigsberg, W., personal communication). By similar isolation, a second peptide was obtained from unsuccinylated catalytic subunit, containing only the pyridoxylated lysine, which corresponds to Lys-80. Derivatives of catalytic subunit containing an average of either one, two, or three pyridoxamine-P moieties per trimer have been prepared by reduction. These species, which retain catalytic activity in proportion to their unmodified active sites, were recombined with regulatory subunit to prepare partially modified derivatives of native aspartate transcarbamylase. At pH 8, fluorescence emission bands were observed at 340 nm, due to aromatic amino acids in the protein, and at 395 nm, due to the pyridoxamine-P moiety. Upon excitation at 280 nm energy transfer from protein to pyridoxamine-P was approximately 15%. The properties of the probe were used to study changes accompanying the binding of substrates and inhibitors. The effects of CTP and ATP were small. With the transition state analog N-(phosphonacetyl)-L-aspartate (PALA) or the substrate carbamyl-P, two types of response were observed. Derivatives of catalytic subunit and native enzyme which contain some unmodified sites and hence retain partial catalytic activity gave large increases in fluorescence at 395 nm. However, fully modified inactive derivatives gave much smaller increases. A derivative of native enzyme containing one triply modified and one unmodified catalytic subunit behaved like the other partially modified species. These results indicate that there is communication among the active sites of different catalytic trimers in modified native enzyme, as well as among active sites within the same modified catalytic trimer. The increases in fluorescence result from a red shift of the absorption maximum of the pyridoxamine-P moiety from 315 to 325 nm, which increases the absorbance at the excitation wavelength for fluorescence. At pH 7, the absorption spectrum is already shifted and, consequently, the binding of PALA and carbamyl-P has little effect on the fluorescence. Therefore, the binding of these compounds at pH 8.0 must cause a structural change in the protein, which in turn causes protonation of a group in the modified active sites, altering the spectral properties.     

In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. 3. Catalytic and regulatory properties of carbamylphosphate synthetase: channeling of carbamylphosphate to aspartate transcarbamylase

The present work reports direct evidence for the channeling of carbamylphosphate from carbamylphosphate synthetase to aspartate transcarbamylase in the multifunctional protein that catalyzes the two first reactions of the pyrimidine pathway in Saccharomyces cerevisiae. This phenomenon is almost certainly related to the previously reported observation that the apparent in situ catalytic mechanism of aspartate transcarbamylase is altered by the association of this enzyme to carbamylphosphate synthetase. As a prerequisite of this investigation, the in situ catalytic and regulatory properties of carbamylphosphate synthetase were studied in the permeabilized cells of a strain that contains the wild-type multifunctional protein but is devoid of the carbamylphosphate synthetase specific for the arginine pathway.     

A 13C tracer method for quantitating de novo pyrimidine biosynthesis in vitro and in vivo

Gas chromatographic/mass spectrometric methods for the measurement of the flux through the de novo pyrimidine biosynthetic pathway by quantitating the incorporation of [13C]bicarbonate and 13CO2 into the uracil nucleotide pool in L1210 tumors are reported. Simultaneous measurements of the incorporation of [13C]bicarbonate and the more commonly used [14C]bicarbonate into uridine of L1210 cells in vitro showed that the two methods were comparable. A modification of the method was applied to in vivo studies where the incorporation of 13CO2 into the uracil nucleotide pool of L1210 tumors in mice was quantitated. The measurements were used to determine changes in the flux through the de novo pyrimidine pathway in animals pretreated with known inhibitors of the pathway. A comparison of control animals with those pretreated with 6-azauridine, acivicin, and pyrazofurin resulted in mean percentage inhibitions of 87, 95, and 94%, respectively. This technique should allow investigation of the respective contributions of salvage and de novo synthesis in the formation of pyrimidines in vivo and the effects of agents designed as enzyme inhibitors of the de novo pathway.