Glutamine utilization in both the arginine-specific and pyrimidine-specific carbamoyl phosphate synthase enzymes of eurospora crassa.

Summary As glutamine-dependent carbamoyl phosphate synthetase (CPS) activity in some organisms is composed of a glutaminase and an ammonium-dependent CPS, CPS^- mutants in Neurospora crassa were examined for glutamine- and ammonium-dependent CPS activities. No evidence was found that the genetic location of these two functions were separable. This is discussed with reference to the close genetic proximity of the CPS_pyr and aspartate carbamoyl-transferase (ACT) structural gene (pyr-3) and the arg-2 gene which appears to specify a subunit responsible for glutamine utilisation in CPS_arg.Supported by Science Research Council Grant B/RG/2981     

Isolation and mapping of fluoropyrimidine-resistant mutants of Neurospora crassa

Summary Growth characteristics of wild type Neurospora crassa on 5-fluorouracil, 5-fluorouridine and 5-fluorodeoxyuridine and methods for selecting mutants resistant to these pyrimidine analogues are described. The mutants are mapped and characterized for resistance to the analogues. Some mutants are found to be allelic with the previously described ud-1 (fdu-1) ad uc-4 genes.Supported by S.R.C. grant GR/A/64655F. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

Genetic and Physiological Aspects of Resistance to 5-Fluoropyrimidines in Saccharomyces cerevisiae

Mutants resistant to 5-fluorouracil, 5-fluorocytosine, and 5-fluorouridine were selected in yeast, and the mechanisms of their resistance were investigated. The investigated mutations map in seven different loci. (i) A mutation at the locus FUI 1 gives specifically resistance to 5-fluorouridine. (ii) Two loci are involved in a specific 5-fluorocytosine resistance: a mutation at locus FCY 1 produces a loss of cytosine deaminase activity; a mutation at locus FCY 2 results in the loss of the activity of a cytosine-specific permease. (iii) A mutation at the locus FUR 4 gives a simultaneous resistance to 5-fluorouracil and to 5-fluorouridine by loss in the activity of the uracil-specific permease. (iv) We found three types of mutants in the locus FUR 1. One is dominant and weakly resistant to 5-fluorouracil, 5-fluorocytosine, and 5-fluorouridine. The two others are recessive and are unable to catalyze one of the steps involved in uracil transformation into uridine 5'-monophosphate; this block-age explains their strong resistance to 5-fluorouracil and 5-fluorocytosine. Of these two mutants, one is resistant to 5-fluorouridine and the other is not. (v) Mutations at locus FUR 2 give resistance to 5-fluorouracil, 5-fluorocytosine, and 5-fluorouridine. These mutations are dominant and lead to a loss in the feedback regulation of the aspartic transcarbamylase activity by uridine triphosphate. (vi) The mutants FUR 3 are resistant to 5-fluorocytosine and 5-fluorouridine. They are dominant and physiologically related to the mutants of the locus FUR 1 but their mechanism of resistance is not understood.     

Elevated uridine nucleotide pools in fluorouracil/fluorouridine resistant mutants ofNocardia lactamdurans

Summary Selection of spontaneous mutants ofNocardia lactamdurans MA2908 for resistance to 5-fluorouracil results in the simultaneous development of resistance to 5-fluorouridine. The resulting mutants fall into four distinct classes based on the amount of uracil accumulating in fermentation broths. An additional characteristic of these mutants is a reduction in the ability to incorporate exogenous uracil into nucleic acids even though transport and conversion to the nucleotide level appears normal. Finally, production of efrotomycin is increased in these mutants in both chemically defined and complex fermentation media to levels equivalent to those of MA4820, the first productivity mutant isolated in a conventional strain improvement program. Resistance development and uracil excretion are adequately explained by an elevation of the intracellular uridine nucleotide pool, in particular UMP. The role of the uridine necleotides in the efrotomycin fermentation is unknown.     

Nucleotide sequence of the pyrimidine specific carbamoyl phosphate synthetase, a part of the yeast multifunctional protein encoded by the URA2 gene

Summary Yeast URA2 encodes a multifunctional carbamoyl phosphate synthetase-aspartate transcarbamylase of 220,000 molecular weight. We determined the nucleotide sequence of the 5 proximal part of the gene which is responsible for the glutamine amide transfer function of the carbamoyl phosphate synthetase activity. Alignment of the enzyme sequence derived from URA2 with sequences from Escherichia coli carA carB and yeast arginine-specific CPA1 CPA2 indicates that monofunctional and bifunctional carbamoyl phosphate synthetases are probably homologous. The URA2-derived enzyme organization is NH₂-carbamoyl phosphate synthetase-aspartate transcarbamylase-CO₂H.     

Isolation and characterization of mutants of mesophilic methanogenic bacteria resistant to analogues of DNA bases and nucleosides

Spontaneous mutants of mesophilic Methanobacterium, Methanobrevibacter and Methanosarcina species resistant to 6-mercaptopurine, 5-fluorouracil, 8-azaguanine, 6-azauracil or 5-fluorodeoxyuridine were isolated. Low level resistant mutants were unstable but highly resistant strains (resistance factor greater than 10-fold) were stable and showed growth characteristics comparable to the parent. Wild type strains showed linear uptake of hypoxanthine and uracil into cells, but guanine uptake was only detected in Methanosarcina mazei. 6-Mercaptopurine-resistant clones of Methanobacterium and Methanobrevibacter species and 8-azaguanine-resistant clones of Methanosarcina mazei showed reduced uptake of hypoxanthine and guanine respectively, but no evidence for altered permeability of 5-fluoro-and 6-azauracil-resistant strains to uracil was obtained. Double resistant mutants of Methanobacterium sp. strain FR-2 were characterised. Although these generally exhibited reduced specific growth rates, several were selected which showed similar growth to the parent.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Federal Republic of Germany - MJC minimum inhibitory concentration - cfu colony forming unit - MP 6-mercaptopurine - FU 5-fluorouracil - FDU 5-fluorodeoxyuridine - AG 8-azaguanine - AU 6-azauracil - DA l-deazaadenosine     

Mutations affecting uridine monophosphate pyrophosphorylase or the argR gene in Escherichia coli. Effects on carbamoyl phosphate and pyrimidine biosynthesis and on uracil uptake

Summary In the course of experiments directed towards the isolation of mutants of Escherichia coli K12 with altered regulation of the synthesis of carbamoylphosphate synthetase, two types of mutations were found to affect the cumulative repression of this enzyme by arginine and uracil. Alteraction of the arginine pathway regulatory gene, argR, was shown to reduce the repressibility of the enzyme by both end products while mutations affecting uridine monophosphate pyrophosphorylase (upp) besides affecting uracil uptake preclude enzyme repression by uracil or cytosine in the biosynthesis of carbamoylphosphate and the pyrimidines. The upp mutations were located on the chromosome near the gua operon. Mutations previously designated as uraP are shown to belong to this class.The relation that could exist between the loss of uridine monophosphate pyrophosphorylase and the impairment of uracil uptake is discussed.A new method for isolating argR mutants in arginine-less strains is described.     

Arginine-specific carbamoyl phosphate metabolism in mitochondria of Neurospora crassa. Channeling and control by arginine

Citrulline is synthesized in mitochondria of Neurospora crassa from ornithine and carbamoyl phosphate. In mycelia grown in minimal medium, carbamoyl phosphate limits citrulline (and arginine) synthesis. Addition of arginine to such cultures reduces the availability of intramitochondrial ornithine, and ornithine then limits citrulline synthesis. We have found that for some time after addition of excess arginine, carbamoyl phosphate synthesis continued. Very little of this carbamoyl phosphate escaped the mitochondrion to be used in the pyrimidine pathway in the nucleus. Instead, mitochondrial carbamoyl phosphate accumulated over 40-fold and turned over rapidly. This was true in ornithine- or ornithine carbamoyltransferase-deficient mutants and in normal mycelia during feedback inhibition of ornithine synthesis. The data suggest that the rate of carbamoyl phosphate synthesis is dependent to a large extent upon the specific activity of the slowly and incompletely repressible synthetic enzyme, carbamoyl-phosphate synthetase A. In keeping with this conclusion, we found that when carbamoyl-phosphate synthetase A was repressed 2-10-fold by growth of mycelia in arginine, carbamoyl phosphate was still synthesized in excess of that used for residual citrulline synthesis. Again, only a small fraction of the excess carbamoyl phosphate could be accounted for by diversion to the pyrimidine pathway. The continued synthesis and turnover of carbamoyl phosphate in mitochondria of arginine-grown cells may allow rapid resumption of citrulline formation after external arginine disappears and no longer exerts negative control on ornithine biosynthesis.     

Factors accelerating pyrimidine production in <Emphasis Type="Italic">Deinococcus radiophilus</Emphasis>

In studying the pyrimidine synthesising pathway in Deinococcus radiophilus two instances of anomalous behaviour were observed. One was the strikingly different results obtained for two types of assay for carbamoyl phosphate synthetase. Both depend on the fixation of ¹⁴C from the substrate bicarbonate to give radioactive products. In the coupled assay the carbamoyl phosphate product of the enzyme is converted to carbamoyl aspartate in the presence of aspartate and aspartate transcarbamoylase. In the direct assay aspartate is omitted from the reaction mixture and the carbamoyl phosphate is converted to urea. It was found that the radioactive counts in the direct assay were about 5% of those measured in the coupled assay. The second anomaly was that omission of glutamine from both assay mixtures had no significant effect on the fixation of radioactive carbon. These results suggested that aspartate amino-N could be the source of nitrogen for glutamine synthesis by a substrate-channelled pathway which delivered glutamine to carbamoyl phosphate synthetase, and that externally added glutamine could not access its binding site on the enzyme.     

Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii

New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage, suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of uridine 5′-monophosphate (UMP) and thymidine 5′-monophosphate (dTMP) pools, thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII-deficient mutants, restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within, or proximal to, the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition, several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development.     

Biochemical and genetic basis of the response to 5-fluorouracil in Drosophila melanogaster

Mutant strains sensitive and resistant to the drug 5-fluorouracil (FU) have been isolated from the wild-type Pac strain of Drosophila melanogaster. The resistant strain, termed flu^r, is resistant to at least 0.0035%FU (2.7 × 10^–4 m) in the food media and exhibits cross-resistance to 5-fluorodeoxyuridine (FUdR) but not to 5-fluorouridine (FUR). The sensitive strain termed flu ^S , exhibits over 90% mortality on 0.0008% FU (6 × 10^–5 m). Genetic analysis indicates that the flu gene is located on the third chromosome, which agrees with results of previous workers. An analysis of the enzyme thymidylate synthetase from the selected sensitive and resistant strains indicates that the resistant strain enzyme possesses an elevated specific activity. Levels 4 times that of the sensitive strain were observed when the enzymes were assayed at 20 C. This increase is apparently not due to induction by FU in the food media. It is suggested that the enzyme thymidylate synthetase may be involved in the resistance process.     

Breeding of a 5-Fluorouridine-resistant Mutant with Increased Cellulose Production from Acetobacter xylinum subsp. nonacetoxidans

UDP-glucose (UDP-G), the direct precursor of cellulose, is known to be produced from UTP and glucose-1-phosphate. In an attempt to increase UTP biosynthesis, 5-fluorouridine (5-FUR: a pyrimidine analog)-resistant mutants were obtained using Acetobacter xylinum subsp. nonacetoxidans 757 as the parent strain. One of the 5-FUR-resistant mutants, FUR-35, showed about 40% higher cellulose productivion compared to the parent strain. Intracellular levels of UTP and UDP-G in FUR-35 was found to be higher than those in the parent strain. The carbamyl phosphate synthetase II (CPS) activity of FUR-35 was higher than that of the parent strain and the feedback inhibition of CPS by UTP in FUR-35 had been released compared with that in the parent strain. These results suggest that the increased cellulose production of FUR-35 was attributable to its higher of intracellular UDP-G level resulting from increased UTP biosynthesis.     

Galactose metabolism inErwinia carotovora Subsp.carotovora

The pyrimidine analogue 5-fluorouracil was shown to be a potent inhibitor of the growth ofMethanobacterium thermoautotrophicum strain Marburg (50% inhibition of growth at 1 g ml^–1). The nucleoside, 5-fluorodeoxyuridine, also inhibited growth, but the nucleotide 5-fluorodeoxyuridylate did not inhibit, nor did 5-fluorocytosine. Several nucleobases and nucleosides were used as potential antagonists of fluorouracil and fluorodeoxyuridine. Of these, only uracil in excess over fluorouracil relieved the inhibition of growth. These results imply that a pyrimidine salvage pathway is present inM. thermoautotrophicum. 5-Fluorouracil does not inhibit methane production. Although treated cultures produced less methane than did controls, more than twice as much methane was synthesized per cell. This result suggests that methanogenesis is uncoupled from growth by 5-fluorouracil.     

Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5′-monophosphate (UMP) from uridine and adenine 5′-triphosphate (ATP) and from uracil and phosphoribosyl-α-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprt1 mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.     

The stimulation of the mitochondrial respiration by citrulline synthesis.

1. The influence of ammonia and ornithine on the oxygen uptake and the formation of citrulline was investigated with isolated rat liver mitochondria. The experiments were performed in a cytosol-like saline medium at 38 degrees C. 2. Under these conditions an increase of the respiration rate by ammonia and ornithine was observed, but a small response to external ADP, only. The missing stimulation by ADP was due to a partial inhibition of the respiratory chain by traces of zinc (approximately 1 microM) present in the medium. This inhibition was only detected at low concentrations of mitochondria. 3. For activation of respiration by ammonia plus ornithine two different processes were responsible: (i) chelation of the inhibiting zinc by ornithine, which could be prevented by EDTA; (ii) ADP production in the matrix space during formation of carbamoyl phosphate, which could be prevented by oligomycin but not by carboxyatractyloside. 4. This stimulus of the carbamoyl phosphate formation and of the equivalent citrulline synthesis on the mitochondrial respiration ran to 12% of that increase caused by phosphorylation of external ADP. The maximum rate of citrulline formation was limited by the activity of carbamoyl phosphate synthetase. 5. Added ADP suppresses the production of citrulline probably by the exchange of extramitochondrial ADP versus intramitochondrial ATP. The data suggest a common adenine nucleotide pool delivering ATP to the adenine nucleotide translocase as well as to the carbamoyl phosphate synthetase.     

Properties of pea seedling uracil phosphoribosyltransferase and its distribution in other plants

A uracil phosphoribosyltransferase (UMP-pyrophosphorylase) was found in several angiosperms and was partially purified from epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings. Its pH optimum was about 8.5; its required approximately 0.3 mm MgCl₂ for maximum activity but was inhibited by MnCl₂; its molecular weight determined by chromatography on Sephadex G-150 columns was approximately 100,000; its K_m values for uracil and 5-phosphorylribose 1-pyrophosphate were 0.7 μm and 11 μm; and it was partially resolved from a similar phosphoribosyltransferase converting orotic acid to orotodine 5′-phosphate. Enzyme fractions containing both uracil phosphoribosyl transferase and orotate phosphoribosyltransferase converted 6-azauracil and 5-fluorouracil to products with chromatographic properties of 6-azauradine 5′-phosphate and 5-fluorouridine 5′-phosphate. Uracil phosphoribosyltransferase probably functions in salvage of uracil for synthesis of pyrimidine nucleotides.     

Restricted passage of reaction intermediates through the ammonia tunnel of carbamoyl phosphate synthetase

The x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit. The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit. A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channeled through the ammonia tunnel. NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine. The kinetic results indicate that hydroxylamine, derived from the hydrolysis of gamma-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit. Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes. The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase.     

Deconstruction of the catalytic array within the amidotransferase subunit of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase subunit, and a small amidotransferase subunit, which belongs to the Triad family of glutamine amidotransferases. Previous studies have established that the reaction mechanism of the small subunit proceeds through the formation of a gamma-glutamyl thioester with Cys-269. The roles in the hydrolysis of glutamine played by the conserved residues, Glu-355, Ser-47, Lys-202, and Gln-273, were determined by mutagenesis. In the X-ray crystal structure of the H353N mutant, Ser-47 and Gln-273 interact with the gamma-glutamyl thioester intermediate [Thoden, J. B., Miran, S. G., Phillips, J. C., Howard, A. J., Raushel, F. M., and Holden, H. M. (1998) Biochemistry 37, 8825-8831]. The mutants E355D and E355A have elevated values of K(m) for glutamine, but the overall carbamoyl phosphate synthesis reaction is unperturbed. E355Q does not significantly affect the bicarbonate-dependent ATPase or glutaminase partial reactions. However, this mutation almost completely uncouples the two partial reactions such that no carbamoyl phosphate is produced. The partial recovery of carbamoyl phosphate synthesis activity in the double mutant E355Q/K202M argues that the loss of activity in E355Q is at least partly due to additional interactions between Gln-355 and Lys-202 in E355Q. The mutants S47A and Q273A have elevated K(m) values for glutamine while the V(max) values are comparable to that of the wild-type enzyme. It is concluded that contrary to the original proposal for the catalytic triad, Glu-355 is not an essential residue for catalysis. The results are consistent with Ser-47 and Gln-273 playing significant roles in the binding of glutamine.     

Effects of fluorinated pyrimidines on the growth of excised pea embryos

The effects of some fluorinated pyrimidines on the growth ofexcised pea embryos {Pisum sativum var. Alaska) in sterile culturewere studied. Even the lowest concentrations of the compoundstested inhibited growth in length of the embryos. In order ofdecreasing activity, the compounds tested were : 5-fluorodeoxyuridine,5-fluorodeoxycytidine, 5-fluorouridine, 5-fluoroorotic acidand 5-fluorouracil. Inhibition of growth in length of the rootprimordia was found to be mainly due to inhibition of cell divisionwith no effect on cell elongation. Reversal of fluoropyrimidineinduced inhibition of growth by pyrimidine bases and their relatedmetabolites indicated that the analogues primarily inhibitedDNA synthesis. ¹Part of a thesis submitted by the senior author for the degreeof M.Sc. of the University of Malaya ²Present address: Department of Botany, University College ofWales, Aberystwyth, U.K. (Received October 27, 1969; )