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1
Ibrahim, Mohamed M. "Pyrimidine Metabolism in Rhizobium: Physiological Aspects of Pyrimidine Salvage." Thesis, University of North Texas, 1989. https://digital.library.unt.edu/ark:/67531/metadc330907/.
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The objective of this research was to study the pyrimidine salvage pathways of Rhizobium. Three approaches were used to define the pyrimidine salvage pathways operative in two species of Rhizobium, R. meliloti and R. leguminosarum . The first approach was to ascertain the pyrimidine bases and nucleosides that could satisfy the pyrimidine requirement of pyrimidine auxotrophs. Uracil, cytosine, uridine or cytidine all satisfied the absolute pyrimidine requirement. The second approach was to select for mutants resistant to 5-fluoropyrimidine analogues which block known steps in the interconversion of the pyrimidine bases and nucleosides. Mutants resistant to 5-fluorouracil lacked the enzyme uracil phosphoribosyltransferase (upp ) and could no longer use uracil to satisfy their pyrimidine requirement. Mutants resistant to 5-fluorocytosine, while remaining sensitive to 5- fluorouracil, lacked cytosine deaminase (cod) and thus could no longer use cytosine to satisfy their pyrimidine auxotrophy. The third approach used a reversed phase HPLC column to identify the products that accumulated when cytidine, uridine or cytosine was incubated with cell extracts of wild type and analogue resistant mutants of Rhizobium. When cytidine was incubated with cell extracts of Rhizobium wild type, uridine, uracil and cytosine were produced. This Indicated that Rhizobium had an active cytidine deaminase (cdd) and either uridine phosphorylase or uridine hydrolase. By dialyzing the extract and reincubating it with cytidine, uridine and uracil still appeared. This proved that it was a hydrolase ( nuh ) rather than a phosphorylase that degraded the nucleoside. Thus, Rhizobium was found to contain an active cytidine deaminase and cytosine deaminase with no uridine phosphorylase present. The nucleoside hydrolase was active with cytidine, uridine and to a far lesser extent with purines, adenosine and inosine. When high concentrations of cytidine were added to mutants devoid of hydrolase, cytosine was produced from cytidine - 5-monophosphate by the sequential action of uridine ( cytidine ) kinase and nucleoside monophosphate glycosylase. Both ft meliloti and ft leguminosarum had identical salvage pathways.
2
Hughes, Lee E. (Lee Everette). "Pyrimidine Metabolism in Streptomyces griseus." Thesis, University of North Texas, 1994. https://digital.library.unt.edu/ark:/67531/metadc278710/.
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Salvage of pyrimidine nucleosides and bases by S. griseus and the regulation of aspartate transcarbamoylase (ATCase) were studied. The velocity-substrate curve for S. griseus ATCase was hyperbolic for both aspartate and carbamoylphosphate. The enzyme activity was diminished in the presence of ATP, CTP, or UTP. The synthesis of ATCase was repressed in cells grown in the presence of exogenous uracil. The specific activity of cells grown with uracil was 43 percent of that for cells grown in minimal medium only. Maximal ATCase and dihydroorotase activities were found in the same column fraction after size-exclusion chromatography, suggesting that both activities could reside in the same polypeptide. The pyrimidine salvage enzymes cytosine deaminase and uridine phosphorylase were identified in S. griseus using HPLC reversed-phase chromatography.
3
Patel, Monal V. "The regulatory roles of PyrR and Crc in pyrimidine metabolism in Pseudomonas aeruginosa." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc2875/.
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The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a pyrR- strain compared to the isogenic pyrR+ strain. Thus, pyrR is negatively regulated while the other pyr genes (except pyrC) are positively activated by PyrR. That no regulation was seen for pyrC is in keeping with the recent discovery of a second functional pyrC that is not regulated in P. aeruginosa. Gel filtration chromatography shows the PyrR protein exists in a dynamic equilibrium, and it is proposed that PyrR functions as a monomer in activating pyrD, pyrE and pyrF and as a dimeric repressor for pyrR by binding to the inverted repeat. A related study discovered that the catabolite repression control (Crc) protein was indirectly involved in pyr gene regulation, and shown to negatively regulate expression of PyrR at the posttranscriptional level.
4
Black, Duncan Arthur. "Aspects of purine and pyrimidine metabolism." Doctoral thesis, University of Cape Town, 1989. http://hdl.handle.net/11427/26590.
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In Chapter 1 a review of the literature concerning aspects of erythrocyte membrane transport and metabolism, and purine and pyrimidine metabolism is presented. The effects of pH, pO₂ and inorganic phosphate (Pi) on the uptake and metabolism of hypoxanthine by erythrocytes has been studied in Chapter 2. Uptake of hypoxanthine and accumulation of inosine 5'-monophosphate (IMP) were markedly increased at acid pH, high external phosphate concentrations, and low pO₂. Release of accumulated IMP as hypoxanthine occurred at alkaline pH values and low external phosphate concentrations. Conditions favouring IMP accumulation gave rise, in the absence of hypoxanthine, to a corresponding increase in 5'-phosphoribosyl-1-pyrophosphate (PRPP). Intracellular phosphate concentrations were markedly pH dependent and a model is presented whereby hypoxanthine uptake and release are controlled by intracellular concentrations of inorganic phosphate and 2,3- bisphosphoglycerate (2,3-DPG). These allosteric effectors influence, in opposing ways, two enzymes governing IMP accumulation, namely PRPP synthetase and 5'-nucleotidase. These metabolic properties suggest that the erythrocyte could play a role in the removal of hypoxanthine from anoxic tissue. In Chapter 3 the kinetics and mechanism of transport of orotate across the human erythrocyte membrane and the effect of pH and inorganic phosphate on its metabolism (in the erythrocyte) have been studied. It has been shown that orotate enters erythrocytes with non-saturable kinetics and with a capacity of 190 μmoles/1 packed cells/min at a concentration of 4-6 mmolar. The presence of competition for transport by a number of anions and the lack of competition by uridine is indicative of transport by a general anion transporter, with the ability for concentrative uptake in the absence of other external anions being compatible with transport via a ping-pong mechanism. Inhibition of transport by the specific band 3 inhibitors DIDS and CHCA confirm that transport is via the band 3 anion transporter. This explains the lack of significant uptake of orotate by most differentiated tissues which lack the intact band 3 protein. However, the demonstration of band 3 in rat hepatocytes (Cheng and Levy, 1980) provides a mechanism for the orotate transport which has been observed in liver (Handschumacher and Coleridge, 1979). Changes in pH and inorganic phosphate (Pi) concentrations have been shown to have marked effects on the relative quantities of metabolic products produced by the erythrocyte from orotate. There was an increase in orotate metabolised with increasing Pi, an effect augmented by lowering the pH, and most easily explained by the allosteric activation of PRPP synthetase by Pi. The increase in UTP levels with decreasing pH may be the consequence of both increased PRPP availability for the formation of uridine nucleotide from orotate, and decreased conversion of UMP to uridine by pyrimidine 5'-nucleotidase, which is known to be inhibited by phosphate. The accumulation of UDP sugars is optimal at a phosphate concentration of 10 mmolar, which is unexplained but would be compatible with an inhibitory effect of Pi on CTP synthetase. A PRPP wasting cycle at alkaline pH values is proposed to explain the apparent paradox where no PRPP was observed to accumulate in erythrocytes (Chapter 2) at pH values of 7.6 and above in the presence of 10 mmolar phosphate and no added hypoxanthine, yet the metabolism of orotate, which is a PRPP utilising reaction, at alkaline pH values was readily demonstrable here. This (apparent paradox) can be resolved if one assumes that even in the absence of added hypoxanthine and demonstrable intracellular IMP there are sufficient quantities of hypoxanthine and/or IMP to maintain a PRPP wasting cycle at alkaline pH values. The cycle is interrupted at acidic pH values as phosphate levels rise and inhibit 5'-nucleotidase, an effect augmented by the decreasing levels of 2,3-DPG which accompany decreasing pH. This wasting cycle has recently been confirmed by P. Berman (unpublished). The kinetics of orotate uptake by erythrocytes and its eventual release as uridine provides a role for the erythrocyte in the transport and distribution of pyrimidines to peripheral tissues. A model is proposed and involves the de novo production of orotate in the liver. In the next step erythrocytes take up the orotate secreted by the liver into the circulation, convert it into an intermediate buffer store of uridine nucleotides, whose distribution is a function of pH and phosphate concentration, and eventually release it as uridine, which is a readily available form of pyrimidine for utilisation by peripheral nucleated cells. The enhancement of uptake of labelled orotate into nucleic acids of cultured cells is demonstrated here. The degradative half of the cycle proposes that uracil and palanine are the predominant degradative forms of pyrimidines produced by peripheral cells, and their ultimate metabolic fate is complete catabolism in the liver to CO₂ and water. In the final chapter the possible role of the human erythrocyte in the prevention of reperfusion injury has been investigated. The development of a model of renal ischaemia in the rat is described. The ability of human erythrocytes, "primed" by preincubating in acid medium of high Pi concentration and low pO₂, to take up hypoxanthine in a concentrative manner when perfused through ischaemic rat kidney is demonstrated. Attempts to demonstrate improved survival and renal function in rats with "primed" human erythrocytes prior to reperfusion were, however, unsuccessful. It is further demonstrated that "unprimed" human erythrocytes, resident in ischaemic rat kidney for 3 hours, take up hypoxanthine and convert it to IMP. that erythrocytes could play a physiological prevention of reperfusion injury.
5
Scott, Allelia Worrall. "Pyrimidine Nucleoside Metabolism in Pseudomonads and Enteric Bacteria." Thesis, University of North Texas, 1991. https://digital.library.unt.edu/ark:/67531/metadc500941/.
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Metabolic differences in the strategies used for pyrimidine base and nucleoside salvage were studied in the pseudomonads and enteric bacteria. Fluoro--analogs were used to select mutant strains of E. coli, S. typhimurium, P. putida, and P. aeruginosa blocked in one or more of the uracil and uridine salvage enzymes. HPLC analysis of cell-free extracts from wild-type and mutant strains examined the effectiveness of the selections. Evidence was found for cytidine kinase in Pseudomonas and for an activity that converted uracil compounds to cytosine compounds. Using media supplemented with 150 μg of orotic acid per ml, P. putida SOC 1, a Pyr, upp mutant which utilizes orotic acid as a pyrimidine source was isolated for the first time in any study.
6
Farajallah, Azizeh M. "Focused chemical libraries targeting pyrimidine metabolism in Plasmodium falciparum /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/8652.
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Brichta, Dayna Michelle. "Construction of a Pseudomonas aeruginosa Dihydroorotase Mutant and the Discovery of a Novel Link between Pyrimidine Biosynthetic Intermediates and the Ability to Produce Virulence Factors." Thesis, University of North Texas, 2003. https://digital.library.unt.edu/ark:/67531/metadc4344/.
Full textAbstract:
The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, we decided to test the ability of the DHOase mutant strain to produce other virulence factors as well. Here we report that a block in the conversion of carbamoyl aspartate (CAA) to dihydroorotate significantly impairs the ability of P. aeruginosa to affect virulence. We believe that the accumulation of CAA in the cell is the root cause of this observed defect. This research demonstrates a potential role for pyrimidine intermediates in the virulence response of P. aeruginosa and may lead to novel targets for chemotherapy against P. aeruginosa infections.
8
Lee, Yick-Shun. "Pyrimidine Metabolism in Bacteria: Physiological Properties of Nucleoside Hydrolase and Uridine Kinase." Thesis, University of North Texas, 1991. https://digital.library.unt.edu/ark:/67531/metadc798309/.
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9
Kumar, Alan P. "Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida." Thesis, University of North Texas, 2003. https://digital.library.unt.edu/ark:/67531/metadc4362/.
Full textAbstract:
The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to DNA thereby enhancing expression of pyrD, pyrE and pyrF genes. When pyrimidine nucleotides are in excess, the PyrR apoprotein binds to orotate, its co-repressor, to shut down all the pyrimidine genes. Like many positive activators, PyrR is subject to autoregulation and has catalytic activity for uracil phosphoribosyltransferase inducible by orotate.
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Léger, Dominique. "L'aspartate transcarbamylase, enzyme clef de la regulation du metabolisme des pyrimidines." Paris 6, 1987. http://www.theses.fr/1987PA066181.
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Rodriguez, Rodriguez Mauricio. "Pyrimidine nucleotide de novo biosynthesis as a model of metabolic control." Texas A&M University, 2005. http://hdl.handle.net/1969.1/4425.
Full textAbstract:
This manuscript presents a thorough investigation and description of metabolic control
dynamics in vivo and in silico using as a model de novo pyrimidine biosynthesis.
Metabolic networks have been studied intensely for decades, helping develop a detailed
understanding of the way cells carry out their biosynthetic and catabolic functions.
Biochemical reactions have been defined, pathway structures have been proposed,
networks of genetic control have been examined, and mechanisms of enzymatic activity
and regulation have been elucidated. In parallel with these types of traditional
biochemical analysis, there has been increasing interest in engineering cellular
metabolism for commercial and medical applications. Several different mathematical
approaches have been developed to model biochemical pathways by combining
stoichiometric and/or kinetic information with probabilistic analysis, or deciphering the
comparative logic of metabolic networks using genomic-derived data. However, most of
the research performed to date has relied on theoretical analyses and non-dynamic
physiological states. The studies described in this dissertation provide a unique effort
toward combining mathematical analysis with dynamic transition experimental data.
Most importantly these studies emphasize the significance of providing a quantitative framework for understanding metabolic control. The pathway of de novo biosynthesis of
pyrimidines in Escherichia coli provides an ideal model for the study of metabolic
control, as there is extensive documentation available on each gene and enzyme involved
as well as on their corresponding mechanisms of regulation. Biochemical flux through
the pathway was analyzed under dynamic conditions using middle-exponential growth
and steady state cultures. The fluctuations of the biochemical pathway intermediates and
end products transitions were quantified in response to physiological perturbation.
Different growth rates allowed the comparison of rapid versus long-term equilibrium
shifts in metabolic adaptation. Finally, monitoring enzymatic activity levels during
metabolic transitions provided insight into the interaction of genetic and biochemical
mechanisms of regulation. Thus, it was possible to construct a robust mathematical
model that faithfully represented, with a remarkable predictability, the nature of the
metabolic response to specific environmental perturbations. These studies constitute a
significant contribution to the fields of quantitative biochemistry and metabolic control,
which can be extended to other cellular processes as well as different organisms.
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Turan, Y. "Pyrimidine primary and secondary metabolism in plants." Thesis, Swansea University, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.639271.
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In this study, the biosynthesis of albizziine has been elucidated, and a direct precursor relationship shown to exist between uracil and albizziine. This was confirmed by the demonstration that [2-14C]uracil specifically labels C-5 of albizziine. It is concluded that the biosynthetic sequence involves the hydroxylation of uracil to isobarbituric acid, then amination to 5-aminouracil, followed by hydrogenation and ring-opening, to yield albizziine. 2,3-Diaminopropanoic acid was shown to be formed from albizziine by the action of β-ureidopropionase. Thus, the formation of albizziine and 2,3-diaminopropanoic acid represents a further aspect of the interfacing of pyrimidine primary and secondary metabolism through uracil. Lathyrine was shown to be catabolyzed in Lathyrus tingitanus to yield the non-protein amino acid 4-hydroxyhomoarginine, and it was thus confirmed that 4-hydroxyhomoarginine is a catabolite rather than a precursor of lathyrine. 2-Amino-4-carboxypyrimidine, the immediate precursor of the lathyrine ring-system, was shown to be synthesized enzymically from uracil. The relative amount of exogenously supplied uracil diverted into production of the isomeric pyrimidinyl amino acids willardiine and isowillardiine in Pisum sativum, and also that diverted into the production of the pyrimidine amino acid lathyrine in Lathyrus tingitanus was determined. Uracil was shown to have a pronounced inhibitory effect on the germination and growth of Phaseolus aureus and Glycine max. As these plants do not produce pyrimidine-derived secondary products, this observation is consistent with the view that production of such compounds is a detoxification mechanism for bioactive pyrimidines.
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Ali, Juma Ahmed Mohmed. "Pyrimidine salvage and metabolism in kinetoplastid parasites." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4664/.
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Pyrimidine uptake has previously been investigated in Trypanosoma brucei procyclics and partly investigated in promastigotes of Leishmania major; however, no such study has been performed using bloodstream forms of Trypanosoma or promastigotes of Leishmania. Here we report a comprehensive study of pyrimidine salvage and metabolism in bloodstream forms of Trypanosoma and promastigotes of Leishmania species. In T. b. brucei bloodstream forms, the uptake of 3H-uracil and 3H-tymidine each appeared to be mediated by a single transporter, designated TbU3 and TbT1, respectively. The procyclic uracil transporter,TbU1, has a high affinity for uracil, with a Km value of 0.46 ± 0.09 μM and Vmax of 0.65 ± 0.008 pmol (107cell)-1 s-1. These values were similar for TbU3 (Km = 0.54 ± 0.11 µM; Vmax = 0.14 ± 0.03), but the main differences between TbU1 and TbU3 are their sensitivity to uridine and 4-thiouracil. Thymidine uptake is detectable at 10 μM over a period from 5 to 30 minutes. This uptake was not inhibited by uracil which indicates that TbT1 is a novel thymidine transporter. The uptake of other pyrimidines, including uridine and 2’-deoxyuridine, by BSF are investigated here but these substrates were also transported by TbU3, and no additional pyrimidine transport activities were found. In L. mexicana and L. major, the uptake of 3H-uracil and 3H-uridine was mediated by separate transporters, designated as follows; for uracil uptake LmexU1, LmajU1; and for uridine uptake LmexNT1, LmajNT1 and LmajNT2, respectively. LmexU1 is a uracil transporter with high affinity to uridine and 2’deoxyuridine, and the LmexNT1 is a nucleoside transporter with broad specificity for purine and pyrimidine nucleosides. L. major uracil transporter (LmajU1) has already been reported by others; and here we report that there are also two distinct uridine transporters expressed in L. major. LmajNT1 is a high affinity uridine transporter which is also inhibited by uracil, inosine and adenine; LmajNT2 is low affinity uridine transporter, with very poor affinity for uracil, inosine and adenine. However, both transporters are inhibited by 2’-deoxyuridine, thymidine and adenosine. Several fluorinated pyrimidine analogues were assessed against kinetoplastid cells, the most effective compounds, which displayed EC50 values at micromolar level, are 5-FU, 5F-2’dUrd, 5-FOA (only against T. brucei BSF) and 5F-Urd (only against L. major). We induced resistance to 5-FU, 5-F2’dUrd and 5-FOA by in vitro exposure of Tbb-BSF and promastigotes of L. mexicana and L. major. The resistance was performed by stepwise increase concentration of the drugs. For T. b. brucei BSF, the resistance factors of clonal lines were 131, 825, and 83-fold, respectively. For L. mexicana and L. major, the resistance factor for 5-FU were 147 and 17-fold, and for 5F-2’dUrd were >3500 and 381-fold, respectively. We also measured 3H–pyrimidine uptake in these cell lines; the resistant bloodstream form strains showed no changes in pyrimidine uptake, with one exception, which is a 76% reduction in 5-FU uptake. In contrast, each resistant strain of Leishmania spp had lost its natural pyrimidine transporter. For example, Leishmania cells resistant to 5-FU had lost uracil transport activity, and cells that were resistant to 5F-2’dUrd had lost uridine transport activity. In addition, we identified kinetoplastid genes that appeared to be associated with resistance to fluorinated pyrimidines. Based on these findings, metabolomic analysis of fluorinated pyrimidines in T. b. brucei resistant cell lines was performed in comparison with parental wild-type; for Leishmania species we only investigated the metabolism of fluorinated pyrimidine in wild type cells, as the fluorinated analogues were simply not taken up in the resistant clones. The metabolomic analysis data showed that, in T. b. brucei, 5-fluorouracil and 5-fluoro orotate are incorporated into a large number of metabolites, but likely act through incorporation into RNA. 5F-2’dUrd and 5F-2’dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase after conversion to 5F-dUMP. Cells treated with 5-fluoro-2’deoxyuridine showed an increase of dUMP, which suggest a block in thymidylate synthase or possibly thymidylate kinase. We also present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes. The effect of fluorinated pyrimidine analogues in the two Leishmania species was almost identical. Each of the tested drugs (5-FU, 5F-2’dUrd and 5F-Urd) produced a limited number of fluorinated metabolites, and their common mode of action was inhibition in thymidylate synthase by 5F-dUMP and thymidine kinase by 5F-2’dUrd. Interestingly, we found that the cause of L. mexicana resistance to 5F-Urd was due to the absence of the 5F-2’dUrd metabolite, as a result of the rapid conversion of 5F-2’dUrd to 5F-dUMP; also we suggest that, in L. mexicana, but not in L. major the high affinity salvage of thymidine is sufficient to provide the cells with thymidine deoxynucleotides. It has been found that pyrimidine salvage is not an essential function for Leishmania cells in vitro conditions. However, it is not known whether either, pyrimidine salvage or biosynthesis, or both of these systems are essential to the trypanosomes in vitro and in vivo study. As T. b. brucei bloodstream forms grew unimpeded in vitro in the complete absence of pyrimidines, uptake is clearly not essential. Disruption of the pyrimidine biosynthesis pathway by deletion of the OMPDCase/OPRTase gene resulted in pyrimidine auxotrophic trypanosomes that were unable to grow in the absence of added pyrimidines. The phenotype was rescued by addition of uracil, and to a lesser extent by some pyrimidine nucleosides. Pyrimidine starvation led rapidly to DNA fragmentation. Adaptations to low pyrimidine availability included upregulation of uracil transport capacity and of uridine phosphorylase expression. However, pyrimidine auxotrophic T. brucei were able to establish a high parasitemia in mice. We therefore conclude that pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity and strongly increased sensitivity to fluorinated pyrimidines. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.
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PERIGNON, JEAN-LOUIS. "Metabolisme des pyrimidines dans les cellules lymphoides humaines." Paris 6, 1987. http://www.theses.fr/1987PA066799.
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Afin d'estimer le role du metabolisme intermediaire des pyrimidines dans la reponse immunitaire, la fonction immunitaire de deux malades ayant un deficit enzymatique hereditaire portant sur la voie de synthese de novo des pyrimidines (oroticurie) est etudie. Le metabolisme intermediaire des nucleosides pyrimidiques dans des lymphocytes et lymphoblastes humains fait l'objet d'une deuxieme etude
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Eguae, Samuel Iyamu. "Pyrimidine nucleotide metabolism in Rhizobium meliloti: purification of aspartate transcarbamoylase from a pyrimidine auxotroph." Thesis, University of North Texas, 1990. https://digital.library.unt.edu/ark:/67531/metadc332674/.
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Rhizobium aspartate transcarbamoylase (ATCase; EC 2.1.3.2) was previously believed to be similar to the Pseudomonas ATCase which has been studied extensively. To facilitate the study of the Rhizobium ATCase a pyrimidine-requiring mutant of R. meliloti was isolated and used in the purification of the enzyme.
16
Fasoli, Marco Oldo. "Pyrimidine transport and salvage metabolism in pathogenic Candida." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315023.
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Stewart, John E. B. (John Edward Bakos). "Characterization of Aspartate Transcarbamoylase in the Archaebacterium Methanococcus Jannaschii." Thesis, University of North Texas, 1996. https://digital.library.unt.edu/ark:/67531/metadc935724/.
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Asparate transcarbamoylase catalyzes the first committed step in the de novo synthesis of pyrmidine nucleotides UMP, UDP, UTP, and CTP. The archetype enzyme found in Escherichia coli (310 kDa) exhibits sigmodial substrate binding kinetics with positive control by ATP and negative control with CTP and UTP. The ATCase characterized in this study is from the extreme thermophilic Archaebacterium, Methanococcus jannaschii. The enzyme was very stable at elevated temperatures and possessed activity from 20 degrees Celsius to 90 degrees Celsius. M. Jannaschii ATCase retained 75% of its activity after incubation at 100 degrees Celsius for a period of 90 minutes. No sigmodial allosteric response to substrate for the enzyme was observed. Velocity substrate plots gave Michaelis-Menten (hyperbolic) kinetics. The Km for aspartate was 7 mM at 30 degrees Celsius and the KM for carbamoylphosphate was .125 mM. The enzyme from M. jannaschii had a broad pH response with an optimum above pH 9. Kinetic measurements were significantly affected by changes in pH and temperature. The enzyme catalyzed reaction had an energy of activation of 10,300 calories per mole. ATCase from M. jannaschii was partially purified. The enzyme was shown to have a molecular weight of 110,000 Da., with a subunit molecular weight of 37,000 Da. The enzyme was thus a trimer composed of three identical subunits. The enzyme did not possess any regulatory response and no evidence for a regulatory polypeptide was found, DNA from M. jannaschii did hybridize to probes corresponding to genes for both the catalytic and regulatory subunits from E. coli. Analysis of DNA sequences for the M. jannaschii ATCase genes showed that the gene for the catalytic subunits shares significant homology with the pyrB genes from E. coli, and maximum homology amongst known ATCase genes to pyrB from Bacillus. An unlinked gene homologous to E. coli pyrl encoding the regulatory subunit was identified, though its expression and true function remain uncharacterized.
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Vickrey, John F. (John Fredrick) 1959. "Isolation and Characterization of the Operon Containing Aspartate Transcarbamoylase and Dihydroorotase from Pseudomonas aeruginosa." Thesis, University of North Texas, 1993. https://digital.library.unt.edu/ark:/67531/metadc278859/.
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The Pseudomonas aeruginosa ATCase was cloned and sequenced to determine the correct size, subunit composition and architecture of this pivotal enzyme in pyrimidine biosynthesis. During the course of this work, it was determined that the ATCase of Pseudomonas was not 360,000 Da but rather present in a complex of 484,000 Da consisting of two different polypeptides (36,000 Da and 44,000 Da) with an architecture similar to that of E. coli ATCase, 2(C3):3(r2). However, there was no regulatory polypeptide found in the Pseudomonas ATCase.
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AsFour, Hani. "Effector Response of the Aspartate Transcarbamoylase From Wild Type Pseudomonas Putida and a Mutant with 11 Amino Acids Deleted at the N-terminus of PyrB." Thesis, University of North Texas, 2002. https://digital.library.unt.edu/ark:/67531/metadc3163/.
Full textAbstract:
Like its enteric counterpart, aspartate transcarbamoylase (ATCase) from Pseudomonas putida is a dodecamer of two different polypeptides. Unlike the enterics, the Pseudomonas ATCase lacks regulatory polypeptides but employs instead inactive dihydroorotases for an active dodecamer. Previous work showed that PyrB contains not only the active site but also the effector binding sites for ATP, UTP and CTP at its N-terminus. In this work, 11 amino acids were deleted from the N-terminus of PyrB and the ATCase with the truncated protein was expressed in E. coli pyrB- and purified. The wild type enzyme was similarly treated. Velocity-substrate plots without effectors gave Michaelis-Menten kinetics in all cases. Deleting 11 amino acids did not affect dodecameric assembly but altered effector responses. When carbamoylphosphate was varied, the mutant enzyme was inhibited by UTP while the wild type enzyme was activated 2-fold. When the aspartate was varied, CTP had no effect on the mutant enzyme but strongly inhibited the wild type enzyme.
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Masson, Anne. "Etude genetique, physiologique et biochimique du metabolisme des pyrimidines chez lactobacillus plantarum." Université Louis Pasteur (Strasbourg) (1971-2008), 1992. http://www.theses.fr/1992STR13189.
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Le metabolisme des pyrimidines chez lactobacillus plantarum, bacterie d'interet agroalimentaire, a ete aborde par des methodes physiologiques et genetiques. Des techniques de mutagenese et d'enrichissement en mutants auxotrophes ont ete mises au point et ont permis d'obtenir de 1 a 9% de mutants parmi la population de bacteries. Des mutants auxotrophes pour les purines, l'histidine, la serine, l'uracile, la thymine, la cytidine. . . Ont ete obtenus. Les mutants auxotrophes pour l'uracile ont ete etudies en dosant les 5 enzymes de biosynthese des pyrimidines. Des mutants deficients dans chacune des 5 etapes ont ete caracterises. De plus, en presence d'uracile dans le milieu de croissance, les 5 activites enzymatiques de la souche sauvage sont nulles. Nous avons montre que l'uracile, ou l'ornithine, inhibent la croissance de l. Plantarum. L'addition d'arginine dans le milieu, ou l'anaerobiose, relevent cette inhibition. Au vu de ces resultats, le dosage de la carbamylphosphate synthetase (cpsase), enzyme necessaire a la synthese de l'arginine et de l'uracile, a ete mis au point. Certains mutants qui possedent les 5 activites enzymatiques de la biosynthese des pyrimidines peuvent pousser, en anaerobiose uniquement sur un milieu minimum depourvu d'arginine et d'uracile et ont une activite cpsase tres faible dans un milieu minimum en aerobiose. L'hypothese est donc la suivante: l. Plantarum sauvage possede deux cpsases; une cpsase specifique des pyrimidines (deficience chez ces mutants) et une cpsase specifique de l'arginine et sensible a l'oxygene
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Hammerstein, Heidi Carol. "Isolation of a Pseudomonas aeruginosa Aspartate Transcarbamoylase Mutant and the Investigation of Its Growth Characteristics, Pyrimidine Biosynthetic Enzyme Activities, and Virulence Factor Production." Thesis, University of North Texas, 2004. https://digital.library.unt.edu/ark:/67531/metadc4704/.
Full textAbstract:
The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.
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Fields, Christopher J. "Comparative biochemistry and genetic analysis of nucleoside hydrolase in Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas fluorescens." Thesis, University of North Texas, 2002. https://digital.library.unt.edu/ark:/67531/metadc3290/.
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The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation.
As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The
physiological relevance of this is explained.
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Hassan, H. F. "Purine and pyrimidine metabolism of Leishmania mexicana mexicana and other parasitic protozoa." Thesis, University of Glasgow, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234859.
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Kim, Hyunju. "Multiple Activities of Aspartate Transcarbamoylase in Burkholderia cepacia: Requirement for an Active Dihydroorotase for Assembly into the Dodecameric Holoenzyme." Thesis, University of North Texas, 2010. https://digital.library.unt.edu/ark:/67531/metadc33176/.
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The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway and virulence factor production, motility tests and biofilm assays were conducted using pyrC- mutant. Even though no significant difference in growth rates was observed, there were significant differences between the wild type and mutant in the production of biofilm and virulence factors. This study will help us to understand the structure and regulation of ATCase holoenzyme with DHOase, and facilitate the use of B. cepacia as an applicable bio-tool. Additionally, we can potentially pursue more efficient drug targets for B. cepacia.
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Wehelie, Rahma. "Mycoplasma pyrimidine deoxynucleotide biosynthesis : molecular characterization of a new family flavin-dependent thymidylate synthase /." Uppsala : Dept. of Molecular Biosciences, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/200676.pdf.
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Linscott, Andrea J. (Andrea Jane). "Regulatory Divergence of Aspartate Transcarbamoylase from the Pseudomonads." Thesis, University of North Texas, 1996. https://digital.library.unt.edu/ark:/67531/metadc277625/.
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Aspartate transcarbamoylase (ATCase) was purified from 16 selected bacterial species including existing Pseudomonas species and former species reassigned to new genera. An enormous diversity was seen among the 16 enzymes with each class of ATCase being represented. The smallest class, class C, with a catalytically active homotrimer, at 100 kDa, was found in Bacillus and other Gram positive bacteria. In this report, the ATCases from the Gram negatives, Shewanella putrefaciens and Stenotrophomonas maltophilia were added to class C membership. The enteric bacteria typify class B ATCases at 310 kDa, with a dodecameric structure composed of two catalytic trimers coupled to three regulatory dimers. A key feature of class B ATCases is the dissociability of the holoenzyme into regulatory and catalytic subunits which were enzymatically active. In this report, the ATCase from Pseudomonas indigofera was added to class B ATCases. The largest class, at 480 kDa, class A, contains the fluorescent Pseudomonas including most members of the 16S rRNA homology group I. Two polypeptides are produced from overlapping pyrBC' genes. The former, pyrB, encodes a 34 kDa catalytic polypeptide while pyrC' encodes a 45 kDa dihydroorotase-like polypeptide. Two non active trimers are made from six 34 kDa chains which are cemented by six 45 kDa chains to form the active dodecameric structure. Dissociation of the holoenyzme into its separate active subunits has not been possible. In this report, the ATCases from Comamonas acidovorans and C. testosteroni, were added to the class A enzymes. An even larger class of ATCase than class A at 600 kDa was discovered in Burkholderia cepacia. Stoichiometric measurements predict a dodecamer of six 39 kDa polypeptides and six 60 kDa polypeptides. Unlike other large pseudomonads ATCases, the enzyme from B. cepacia was dissociable into smaller active forms. Both the holoenzyme and its dissociated forms were regulated by nucleotide effectors. A new class of ATCase was proposed for B. cepacia type enzymes.
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Barron, Vincent N. (Vincent Neal). "Comparison of Aspartate Transcarbamoylase and Pyrimidine Salvage in Sporosarcina urea, Sprolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus." Thesis, University of North Texas, 1994. https://digital.library.unt.edu/ark:/67531/metadc278938/.
Full textAbstract:
The enzyme that catalyzes the committed step in pyrimidine biosynthesis, aspartate transcarbamoylase, has been compared in selected endospore-forming organisms and in morphologically similar control organisms. The ATCases and pyrimidine salvage from Sporosarcina ureae, Sporolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus were compared to those of Bacillus subtilis. While the ATCases from Sporosarcina ureae, Sporolactobacillus inulinus, and L. fermentum were found to exhibit characteristics to that of Bacillus with respect to molecular weight and kinetics, M. luteus ATCase was larger at approximately 480 kDa. Furthermore, pyrimidine salvage in Sporosarcina ureae and M. luteus was identical to those of B. subtilis, while pyrimidine salvage of Sporolactobacillus inulinus and L. fermentum resembled that of the pseudomonads.
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BARDOT, VALERIE. "Modifications du metabolisme des purines et des pyrimidines dans les tumeurs humaines, relation avec les desequilibres chromosomiques." Paris 7, 1994. http://www.theses.fr/1994PA077200.
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Afin de preciser la signification des anomalies chromosomiques observees dans les cancers humains, l'hypothese d'une correlation avec les deregulations metaboliques a ete emise. L'analyse cytogenetique des cancers colorectaux a permis de distinguer deux types de cancers et les desequilibres chromosomiques observes dans ces tumeurs suggerent des modifications du metabolisme des pyrimidines. Une bonne correlation a pu etre mise en evidence, d'une part entre les activites thymidine kinase et thymidylate synthase et le nombre de chromosomes portant les genes respectifs, et d'autre part entre ces activites enzymatiques et le type cytogenetique de ces tumeurs, en particulier apres transplantation sur souris athymiques. Pour confirmer cette hypothese, d'autres tumeurs humaines ont ete etudiees, plus particulierement d'origine neuroectodermique comme les gliomes et les melanomes, pour lesquelles les desequilibres chromosomiques suggerent plutot des alterations du metabolisme des purines. Les activites de plusieurs enzymes impliquees dans ce metabolisme ont donc ete mesurees en relation avec les desequilibres chromosomiques observes dans ces tumeurs. Les gliomes et les melanomes sont caracterises par une baisse globale du metabolisme des purines, affectant la voie de recuperation et le catabolisme, comme le suggeraient les pertes chromosomiques frequemment observees dans ces tumeurs. De plus, les gliomes et les melanomes qui ont a la fois une origine embryonnaire et un profil cytogenetique proches, presentent egalement pour les purines, un profil metabolique comparable. Enfin, les neuroblastomes humains sont des tumeurs egalement d'origine neuroectodermique, qui peuvent se repartir en deux groupes en fonction des donnees cliniques et cytogenetiques. Nos resultats ont montre que ces deux groupes de tumeurs, caracterises par des profils chromosomiques distincts, presentaient deux profils metaboliques differents pour les purines et les pyrimidines. L'ensemble de ces travaux montre qu'il existe bien une relation entre le profil de desequilibres chromosomiques et le profil de deviations metaboliques dans ces tumeurs humaines. Toutefois, les alterations chromosomiques n'expliquent pas les modifications d'activites des enzymes impliquees dans le metabolisme des purines, par un effet de dosage genique. Nos resultats mettent egalement en evidence dans les gliomes, que les modifications metaboliques precedent l'apparition des anomalies chromosomiques et semblent en relation avec la progression tumorale, suggerant leur role determinant dans le processus de cancerogenese, en particulier a des stades precoces
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BAILLON, JEAN. "Les enzymes du metabolisme des nucleotides pyrimidiques comme cibles dans la chimiotherapie antitumorale." Paris 6, 1987. http://www.theses.fr/1987PA066118.
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Hongsthong, Apiradee 1970. "Assembly of Pseudomonas putida Aspartate Transcarbamoylase and Possible Roles of the PyrC' Polypeptide in the Folding of the Dodecameric Enzyme." Thesis, University of North Texas, 1999. https://digital.library.unt.edu/ark:/67531/metadc278618/.
Full textAbstract:
Aspartate transcarbamoylase (ATCase) of Pseudomonas putida consists of two different polypeptides, PyrB and PyrC' (Schurr et al, 1995). The role of the PyrC' and the assembly of PyrB and PyrC' have been studied. The ATCase made in vitro of P.putida PyrB with P.putida PyrC', and of E.coli PyrB with P.putida PyrC ' were generated under two different conditions, denaturation and renaturation, and untreated. It was found that PyrC' plays a role in the enzymatic regulation by ATP, CTP and UTP. In addition to playing a role in substrate binding, the PyrB polypeptide is also involved in effector binding (Kumar et al., manuscript in preparation). The most energetically preferred form of the P.putida WT is a dodecamer with a molecular mass of 480 kDa. The ratio between the PyrB and the PyrC' is 1:1. In studies of nucleotide binding, it was discovered that the P.putida PyrB was phosphorylated by a protein kinase in the cell extract. In the presence of 20 mM EDTA, this phosphorylation was inhibited and the inhibition could be overcome by the addition of divalent cations such as Zn2+ and Mg2+. This result suggested that the phosphorylation reaction required divalent cations. In the CAD complex of eukaryotes, phosphorylations of the CPSase and the linker region between ATCase and DHOase did not occur in the presence of UTP and it was hypothesized (Carrey, 1993) that UTP and phosphorylation(s) regulated the conformational change in the enzyme complex. Therefore, the same idea was approached with P.putida ATCase, where it was found that 1.0 mM UTP inhibited the phosphorylation of PyrB by more than 50%. These results suggested that the regulation of the conformational change of the P.putida ATCase might be similar to that of CAD. Furthermore, peptide mapping for phosphorylation sites was performed on P.putida ATCase WT, WT --11 amino acids and WT --34 amino acids from the N-terminus of the PyrB polypeptide. The results showed that the phosphorylation sites were located on the fragment that contained amino acid number-35 to amino acid number-112 from the N-terminus of the PyrB polypeptide.
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Dill, Michael T. "Characterization of the Aspartate Transcarbamoylase that is Found in the pyrBC Complex of Bordetella Pertussis." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc3057/.
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An aspartate transcarbamoylase (ATCase) gene from Bordetella pertussis was amplified by PCR and ligated into pT-ADV for expression in Escherichia coli. This particular ATCase (pyrB) was an inactive gene found adjacent to an inactive dihydroorotase (DHOase) gene (pyrC'). This experiment was undertaken to determine whether this pyrB gene was capable of expression alone or if it was capable of expression only when cotransformed with a functional pyrC'. When transformed into E. coli TB2 pyrB-, the gene did not produce any ATCase activity. The gene was then co-transformed into E. coli TB2 pyrB- along with a plasmid containing the pyrC' gene from Pseudomonas aeruginosa and assayed for ATCase activity. Negative results were again recorded.
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Higginbotham, Leah. "Aspartate Transcarbamoylase of Aeromonas Hydrophila." Thesis, University of North Texas, 2000. https://digital.library.unt.edu/ark:/67531/metadc5840/.
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This study focused on the enzyme, aspartate transcarbamoylase (ATCase) from A. hydrophila, a Gram-negative bacterium found in fresh water. The molecular mass of the ATCase holoenzyme from A. hydrophila is 310 kDa. The enzyme is likely composed of 6 catalytic polypeptides of 34 kDa each and 6 regulatory polypeptides of 17 kDa each. The velocity-substrate curve for A. hydrophila ATCase is sigmoidal for both aspartate and carbamoylphosphate. The Km for aspartate was the highest to date for an enteric bacterium at 97.18 mM. The Km for carbamoylphosphate was 1.18 mM. When heated to 60 ºC, the specific activity of the enzyme dropped by more than 50 %. When heated to 100 ºC, the enzyme showed no activity. The enzyme's activity was inhibited by ATP, CTP or UTP.
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Hooshdaran, Massoumeh Ziba. "Comparative Biochemistry and Evolution of Aspartate Transcarbamoylase from Diverse Bacteria." Thesis, University of North Texas, 1999. https://digital.library.unt.edu/ark:/67531/metadc500380/.
Full textAbstract:
Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis. Bacterial ATCases are divided into three classes, A, B and C. Class A ATCases are largest at 450-500, are. dodecamers and represented by Pseudomonas ATCase. The overlapping pyrBC' genes encode the Pseudomonases ATCase, which is active only as a 480 kDa dodecamer and requires an inactive pyrC'-encoded DHOase for ATCase activity. ATCase has been studied in two non-pathogenic members of Mycobacterium, M. smegmatis and M. phlei. Their ATCases are dodecamers of molecular weight 480 kDa, composed of six PyrB and six PyrC polypeptides. Unlike the Pseudomonas ATCase, the PyrC polypeptide in these mycobacteria encodes an active DHOase. Moreover, the ATCase: DHOase complex in M. smegmatis is active both as the native 480 kDa and as a 390 kDa complex. The latter lacks two PyrC polypeptides yet retains ATCase activity. The ATCase from M. phlei is similar, except that it is active as the native 480 kDa form but also as 450,410 and 380 kDa forms. These complexes lack one, two, and three PyrC polypeptides, respectively. By contrast,.ATCases from pathogenic mycobacteria are active only at 480 kDa. Mycobacterial ATCases contain active DHOases and accordingly. are placed in class A1 . The class A1 ATCases contain active DHOases while class A2 ATCases contain inactive DHOases. ATCase has also been purified from Burkholderia cepacia and from an E. coli strain in which the cloned pyrB of B. cepacia was expressed. The B. cepacia ATCase has a molecular mass of 550 kDa, with two different polypeptides, PyrB (52 kDa) and PyrC of (39 kDa). The enzyme is active both as the native enzyme at 550 kDa and as smaller molecular forms including 240 kDa and 165 kDa. The ATCase synthesized by the cloned pyrB gene has a molecular weight of 165 kDa composed of three identical PyrB and no PyrC polypeptides. Nucleotide effectors ATP, CTP, and UTP inhibited all forms of enzymes. Because of its size and its activity as a trimer and smaller than native forms, the B. cepacia enzyme is placed in a new class.
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Hermansen, Russell A., Brian K. Mannakee, Wolfgang Knecht, David A. Liberles, and Ryan N. Gutenkunst. "Characterizing selective pressures on the pathway for de novo biosynthesis of pyrimidines in yeast." BioMed Central Ltd, 2015. http://hdl.handle.net/10150/610280.
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BACKGROUND: Selection on proteins is typically measured with the assumption that each protein acts independently. However, selection more likely acts at higher levels of biological organization, requiring an integrative view of protein function. Here, we built a kinetic model for de novo pyrimidine biosynthesis in the yeast Saccharomyces cerevisiae to relate pathway function to selective pressures on individual protein-encoding genes. RESULTS: Gene families across yeast were constructed for each member of the pathway and the ratio of nonsynonymous to synonymous nucleotide substitution rates (dN/dS) was estimated for each enzyme from S. cerevisiae and closely related species. We found a positive relationship between the influence that each enzyme has on pathway function and its selective constraint. CONCLUSIONS: We expect this trend to be locally present for enzymes that have pathway control, but over longer evolutionary timescales we expect that mutation-selection balance may change the enzymes that have pathway control.
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Wei, Shuang. "Modifications du metabolisme des nucleotides en relation avec la differenciation et en reponse a une irradiation dans des cellules tumorales humaines (doctorat : structure et fontionnement des systemes biologiques integres)." Paris 11, 1998. http://www.theses.fr/1998PA114846.
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Schurr, Michael J. (Michael John). "Molecular and Kinetic Characterization of the Aspartate Transcarbamoylase Dihydroorotase Complex in Pseudomonas putida." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc277575/.
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Aerobic Gram negative bacteria such as Pseudomonas putida were reported to possess class A ATCases and to have a M.W. of 360 kD. The nucleotide sequence of the P. putida pyrBC was determined to answer this question once and for all. The expected regulatory gene was not found. It is shown that the P. putida pyrB gene is overlapped by pyrC by 4 bp. The P.putida pyrB is 1005 bp (335 aa) in length and the pyrC is 1275 bp (425 aa) long. Both of these genes complement E. coli mutants with their respective genotypes. Another finding borne out from the sequence is an effector binding site at the N-terminus of pyrB of P. putIda. The binding site shows that effectors compete with carbamoylphosphate for the active site. In this dissertation, it is shown that the ATCase of P.putida is a trimer of M.W. of 109 kD (3 x 36.4 kD) and that the gene encoding pyrB is overlapped by the pyrC gene which encodes DHOase. It is also shown that the pyrBC encoded enzymes copurify as a dodecameric complex with a M.W. of 484 kD.
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Fowler, Michael A. (Michael Allen) 1961. "Characterization of Aspartate Transcarbamoylase and Dihydroorotase in Moraxella Catarrhalis." Thesis, University of North Texas, 1998. https://digital.library.unt.edu/ark:/67531/metadc277709/.
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Bacterial aspartate transcarbamoylases (ATCase's) are divided into three classes that correspond to taxonomic relationships within the bacteria. The opportunistic pathogen Moraxeila catarrhalis has undergone several reclassifications based on traditional microbiological criteria. The previously uncharacterized ATCase from M. catarrhalis was purified to homogeneity and its chemical properties characterized. The ATCase from M. catarrhalis is a class C ATCase with an apparent molecular mass of 480-520 kDa. The M. catarrhalis ATCase is a dodecomer composed of six 35 kDa polypeptides and six 45 kDa polypeptides. The enzyme has an unusually high pH optimum of greater than pH 10. The enzyme exhibited hyperbolic kinetic with a Km for aspartate of 2 mM. A single, separate 78 kDa dihydroorotase from M. catarrhalis was identified and it was not associated with ATCase. These data support the reclassification of M. catarrhalis out of the Neisseriaceae family.
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Cooke, Patrick Alan. "BioInformatics, Phylogenetics, and Aspartate Transcarbamoylase." Thesis, University of North Texas, 2000. https://digital.library.unt.edu/ark:/67531/metadc2580/.
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In this research, the necessity of understanding and using bioinformatics is demonstrated using the enzyme aspartate transcarbamoylase (ATCase) as the model enzyme. The first portion of this research focuses on the use of bioinformatics. A partial sequence of the pyrB gene found in Enterococcus faecalis was submitted to GenBank and was analyzed against the contiguous sequence from its own genome project. A BLAST (Basic Local Alignment Search Tool; Atschul, et al., 1990) was performed in order to hypothesize the remaining portion of the gene from the contiguous sequence. This allowed a global comparison to other known aspartate transcarbamoylases (ATCases) and once deduced, a translation of the sequence gave the stop codon and thus the complete sequence of the open reading frame. When this was complete, upstream and downstream primers were designed in order to amplify the gene from genomic DNA. The amplified product was then sequenced and used later in phylogenetic analyses concerning the evolution of ATCase. The second portion of this research involves taking multiple ATCase nucleotide sequences and performing phenetic and phylogenetic analyses of the archaea and eubacter families. From these analyses, ancestral relationships which dictate both structure and function were extrapolated from the data and discussed.
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Patel, Seema R. "A Study of the Pyrimidine Biosynthesis Pathway and its Regulation in Two Distinct Organisms: Methanococcus jannaschii and Pseudomonas aeruginosa." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc3038/.
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Methanococcus jannaschii is a thermophilic methane producing archaebacterium. In this organism genes encoding the aspartate transcarbamoylase (ATCase) catalytic (PyrB) and regulatory (PyrI) polypeptides were found. Unlike Escherichia coli where the above genes are expressed from a biscistronic operon the two genes in M. jannaschii are separated by 200-kb stretch of genome. Previous researchers have not been able to show regulation of the M. jannaschii enzyme by the nucleotide effectors ATP, CTP and UTP. In this research project we have genetically manipulated the M. jannaschii pyrI gene and have been able to assemble a 310 kDa E. coli like enzyme. By using the second methionine in the sequence we have shown that the enzyme from this organism can assemble into a 310 kDa enzyme and that this enzyme is activated by ATP, CTP and inhibited by UTP. Thus strongly suggesting that the second methionine is the real start of the gene. The regulation of the biosynthetic pathway in Pseudomoans aeruginosa has previously been impossible to study due to the lack of CTP synthase (pyrG) mutants. By incorporating a functional uridine (cytidine) kinase gene from E. coli it has been possible to isolate a pyrG mutant. In this novel mutant we have been able to independently manipulate the nucleotide pools and study its effects on the enzymes in the biosynthetic pathway. The enzyme asapartate transcarbamoylase was repressed 5-fold when exogenous uridine was high and cytidine was low. The enzyme dihydroorotate was repressed 9-fold when uridine was high. These results suggest that a uridine compound may be the primary repressing metabolite for the enzymes encoded by pyrB and pyrC. This is the first study to be done with the proper necessary mutants in the biosynthetic pathway of P aeruginosa. In the past it has been impossible to vary the internal UTP and CTP pools in this organism.
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HORVATH, PHILIPPE. "Dynamique, evolution et expression de genomes de bacteries lactiques : cas du metabolisme des pyrimidines chez lactobacillus plantarum ccm 1904." Université Louis Pasteur (Strasbourg) (1971-2008), 2000. http://www.theses.fr/2000STR13093.
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Lactobacillus plantarum est une bacterie lactique utilisee pour diverses fermentations dans l'industrie agro-alimentaire. Malgre la grande taille de son genome, la souche ccm 1904 est auxotrophe pour de nombreux acides amines et vitamines ; elle est cependant prototrophe pour les pyrimidines. Par sa grande diversite d'organisations genomiques, le metabolisme des pyrimidines constitue un modele interessant pour l'etude de la dynamique et de l'evolution des genomes. Chez plusieurs bacteries gram positives, les genes intervenant dans la biosynthese de novo des pyrimidines sont organises en un grand operon pyrr-p-b-c-aa-ab-k-db-f-e. Mais l'operon pyr de l. Plantarum est incomplet : pyrr-b-c-aa-ab-db-f-e. Par ailleurs, les bacteries lactiques possedent un second gene pyrd distinct (pyrda) ; pyrda et pyrdb codent chacun une dihydro-orotate deshydrogenase fonctionnelle. Les travaux realises au cours de cette these ont permis de mettre en evidence plusieurs originalites propres a l. Plantarum : - l'existence d'un gene pyrd unique, de type b. La construction d'un mutant delete du gene pyrdb par mutagenese dirigee par pcr a conduit a l'obtention d'une souche auxotrophe pour les pyrimidines. Le gene pyrda aurait ete perdu par adaptation a de nouvelles niches ecologiques ou sa presence n'est pas indispensable ; - l'absence du gene pyrk codant une sous-unite de la dihydro-orotate deshydrogenase b. Son absence pourrait etre liee a la non-utilisation du fer par les lactobacilles ; - le groupement, ailleurs sur le chromosome, des genes upp (uracile phosphoribosyltransferase) et pyrp (uracile permease) intervenant dans la voie de sauvetage de l'uracile. Cette situation pourrait correspondre a un deplacement recent du gene pyrp hors de l'operon pyr. De plus, l'etude de mutants de regulation construits par mutagenese dirigee par pcr a permis de montrer que l'expression de l'operon pyr est regulee par attenuation transcriptionnelle mediee par la proteine pyrr.
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Barbosa, Sara Isabel Cadinha. "Compostos que interferem no metabolismo dos purina- e pirimidina-nucleótidos: utilização como agentes terapêuticos." Master's thesis, [s.n.], 2015. http://hdl.handle.net/10284/5160.
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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências FarmacêuticasO conteúdo deste trabalho será desenvolvido em dois temas principais, um referente à utilização de compostos que interferem no metabolismo dos purina- e pirimidinanucleótidos como agentes antineoplásicos e outro referente à sua utilização como agentes antivirais. A síntese dos nucleótidos envolve a construção de ácidos nucleicos e a inserção dos derivados de nucleótidos noutras vias bioquímicas, sendo responsável por inúmeras funções do metabolismo celular. Existem patologias que envolvem enzimas essenciais do metabolismo dos nucleótidos, o que levou à síntese de novos fármacos. As doenças oncológicas continuam a matar milhares de pessoas e um tratamento eficaz e com sucesso tem sido um desafio. O mesmo se passa com algumas infeções virais, nomeadamente infeções provocadas pelo HIV. Para contornar os obstáculos enfrentados na terapia destas doenças têm sido usados análogos de nucleótidos e/ou nucleósidos como agentes terapêuticos. Estes têm o propósito de inibir a síntese de novo dos nucleótidos em determinadas etapas, estando envolvidos na replicação e síntese do RNA e DNA nas células em divisão. Atuam por inibição específica de enzimas no metabolismo dos nucleótidos/nucleósidos ou ainda por incorporação no DNA ou no RNA. This study will be developed into two main subjects; one related to the use of compounds which interfere with the metabolism of purine- and pyrimidine- nucleotides as antineoplastic agents; another related to their use as antiviral agents. The nucleotides’ synthesis involves the construction of nucleic acids and the introduction of the nucleotides’ derivatives into other biochemical pathways and it is responsible for numerous functions of cellular metabolism. There are pathologies involving key enzymes from the nucleotides’ metabolism, which led to the synthesis of new drugs. Cancer is a disease that continues killing thousands of people, an effective and successful treatment has been a challenge. The same happens with some viral infections, mainly infections caused by HIV. To overcome the obstacles faced in the therapy of these diseases it has been used nucleotide and/or nucleoside analogues as therapeutic agents. These agents have the purpose of inhibiting the de novo nucleotide synthesis in certain steps, by being involved in RNA and DNA replication and synthesis in dividing cells. They act by specific enzymes inhibition in nucleotide/nucleoside metabolism and by incorporation into DNA or RNA.
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Stawska, Agnieszka A. "Purification of Aspartate Transcarbamoylase from Moraxella (Branhamella) catarrhalis." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc2864/.
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The enzyme, aspartate transcarbamoylase (ATCase) from Moraxella (Branhamella) catarrhalis, has been purified. The holoenzyme has a molecular mass of approximately 510kDa, harbors predominantly positive charges and is hydrophobic in nature. The holoenzyme possesses two subunits, a smaller one of 40 kDa and a larger one of 45 kDa. A third polypeptide has been found to contribute to the overall enzymatic activity, having an approximate mass of 55 kDa. The ATCase purification included the generation of cell-free extract, streptomycin sulfate cut, 60 °C heat step, ammonium sulfate cut, dialysis and ion, gel-filtration and hydrophobic interaction chromatography. The enzyme's performance throughout purification steps was analyzed on activity and SDS-PAGE gradient gels. Its enzymatic, specific activities, yield and fold purification, were also determined.
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Liu, Haiyan 1966. "Attenuation of Escherichia Coli Aspartate Transcarbamoylase Expressed in Pseudomonas Aeruginosa Mutant and Wild Type Strains." Thesis, University of North Texas, 1994. https://digital.library.unt.edu/ark:/67531/metadc279106/.
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No apparent repression of pyr gene expression in Pseudomonas aeruginosa is observed upon addition of exogenous pyrimidines to the growth medium. Upon introduction of the subcloned Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) into a P. aeruginosa pyrB mutant strain, repression was observed in response to exogenously fed pyrimidine compounds. The results proved that it is possible to bring about changes in pyrimidine nucleotide pool levels and changes in transcriptional regulation of gene expression as a result. Thus, the lack of regulatory control in P. aeruginosa pyr gene expression is not due to an inability to take up and incorporate pyrimidine compounds into metabolic pools, or to an inability of the RNA polymerase to respond to regulatory sequences in the DNA but is probably due to a lack of specific regulatory signals in the promoter of the genes themselves.
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Spiegel, Erin Kathleen. "Psychomotor deficits in mice transgenic for a mutant adenylosuccinate lyase associated with autism in humans /." Connect to full text via ProQuest. IP filtered, 2006.
Find full textAbstract:
Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006.Typescript. Includes bibliographical references (leaves 127-143). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Boussiengui-Boussiengui, Gino. "Analysis of the role of relative nucleotide concentrations on the regulation of carbohydrate in higher plants." Thesis, Stellenbosch : Stellenbosch University, 2010. http://hdl.handle.net/10019.1/5473.
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Thesis (PhD (Genetics))--Stellenbosch University, 2010.ENGLISH ABSTRACT: The current understanding of the regulation of carbohydrate accumulation is still under investigation despite the tremendous work done in this subject. Purine and pyrimidine nucleotides have been implicated in many biochemical processes in plants. Amongst others, they are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products such as sucrose, polysaccharides, phospholipids, as well as secondary products. With the aim of placing adenine and uridine nucleotides in the context of sucrose and starch metabolism and carbon partitioning in higher plant, we have investigated the transcripts, enzymes and metabolites in carbohydrate metabolism and both de novo and salvage of purine and pyrimidine nucleotides in both sugarcane and tobacco tissues. For that purpose, adenylate kinase (ADK) and UMP synthase were chosen for silencing and over expression as they are rate limiting steps of de novo adenine and uridine nucleotides biosynthesis, respectively. Sugarcane with repressed ADK activity showed significant increase in both the starch and adenylate pools. Increase in starch content was highly correlated with reduced ADK activity. As a result of decreased ADK activity, the salvage pathway was up regulated via the increased activity of both adenosine kinase (AK) and adenine phosphoribosyl transferase (APRTase) which positively correlated with increase in adenine nucleotide contents. In addition hexose phosphates and ADP glucose, the committed substrate for starch biosynthesis positively correlated with changes in starch content. A high ratio of ATP/ADP was observed in all transgenic lines compared with the untransformed wild type and suggested to favour starch synthesis. Over expression of cytosolic ADK in tobacco demonstrated an expression of the enzyme where 2/3 of the total activity was in the direction of ADP production. As a result of over expression of ADK, starch content increased in all transgenic plants and positively correlated with changes in the activity of ADK. Despite changes in adenine nucleotide content, the salvage pathway was not activated and no significant changes in both AK and APRTase acivities were found between the transgenic and the untransformed plants. Sucrose synthase (SuSy) activity in breakdown direction positively correlated with changes in starch content suggesting a contribution in the starch accumulation in tobacco plants. In addition the ratio of ATP/ADP was low in all transgenic lines compared with the untransformed wild type. This was in line with the higher content in ADP compare to ATP in all transgenic lines and was supported by the over expression of ADK, and predominantly in the direction of ADP production. Repressed UMP synthase in transgenic sugarcane resulted in increases in sucrose, starch and uridinylate. UDP-glucose, hexose phosphates and uridinylate content positively correlated with changes in sucrose content. Transgenic lines had increased sucrose phosphate synthase (SPS) activity and low activity in SuSy, which suggests alteration of carbon flux toward sucrose. As a result of decreased UMP synthase activity, an up regulation of the salvage pathway was observed and predominantly via increased activity of uridine kinase (UK) which positively correlated with changes in the uridinylate pool. In addition to repressed UMP synthase activity, starch content and adenine nucleotides increased in transgenic lines. Tobacco plants transformed with a cytosolic UMP synthase demonstrated an over expression of the enzyme in all transgenic lines. As a result of over expression of UMP synthase, key metabolites were up regulated, amongst them sucrose. Increase in sucrose content positively correlated with both hexoses and hexose phosphates but not the uridinylate pool. SPS activity positively correlated with increase in sucrose content, and accounted for most of the sucrose synthesized in transgenic lines. Despite the increase in the adenylate pool, no significant changes were observed in starch content. The depletion level of UDP-glucose in all transgenic lines was a mere reflection of the higher activity of UDP glucose pyrophosphorylase (UGPase) in the formation of glucose-1-phosphate. In addition, no salvage pathway was up regulated in transgenic lines.
AFRIKAANSE OPSOMMING: Die huidige beskikbare inligting in verband met die reguleering van koolhidraat akkumulasie word steeds ondersoek ten spyte van die groot hoeveelheid navorsing wat reeds in hierdie verband gedoen is. Purien en pirimidien nukleotide speel ‘n rol in baie biochemiese prosesse in plante. Onder andere is hulle boublokke vir nukleïensuur sintese, ‘n energie bron, voorlopers vir die sintese van primêre produkte soos byvoorbeeld sukrose, polisakkariede, fosfolipiede, asook sekondêre produkte. Met die vooruitsig om adenine- en uridiennukleotide in verband te plaas met sukrose en stysel metabolisme en koolstof afskorting in plante, ondersoek ons hier die transkripte, ensieme en metaboliete in koolhidraat metabolisme in beide de novo en berging van purien en pirimidien nukleotide in suikerriet asook tabak weefsel. Vir hierdie doel is adenilaatkinase (ADK) en UMP-sintase gekies vir uitskakeling en ooruitdrukking, juis omdat hulle tempo vermindering stappe van de novo adenine- en uridiennukleotide biosintese is. Suikerriet met onderdrukte ADK aktiwiteit wys betekenisvolle vermeerdering in beide die stysel en adenilaat poele. Verhoging in styselinhoud was hoogs gekorreleerd met verminderde ADK aktiwiteit. As gevolg van ‘n vermindering in ADK aktiwiteit, is die bergingspad opwaards gereguleer via die vermeerdering van beide adenosienkinase (AK) en adenien-fosforibosieltransferase (APRTase) aktiwiteit wat positief korreleer met die vermeerdering in adeniennukleotied-inhoud. Addisioneel word hexosefosfate en ADP-glukose, die toegewysde substraat vir stysel biosintese, positief gekorreleer met veranderinge in styselinhoud. ‘n Hoë verhouding van ATP/ADP was geobserveer in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe en blyk stysel sintese te begunstig. Ooruitdrukking van sitologiese ADK in tabak demonstreer die uitdrukking van die ensiem waar 2/3 van die totale aktiwiteit in die rigting van ADP produksie was. As ‘n resultaat van ooruitdrukking van ADK, word stysel inhoud vermeerder in alle transgeniese plante en positief gekorreleer met die verandering in die aktiwiteit van ADK. Ten spyte van veranderinge in adeniennukleotide inhoud was die bergingspad nie geaktiveer nie en geen betekenisvolle veranderinge in beide AK en APRTase aktiwiteit was gevind tussen die transgeniese en nie-transgeniese plante nie. Sukrose sintese (SuSy) aktiwiteit tydens afbreking korreleer positief met die veranderinge in stysel inhoud en dui moontlik op ‘n bydrae in die stysel akkumulasie in tabak plante. Verder was die verhouding van ATP/ADP laag in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe. Hierdie bevinding word ondersteun deur die hoër inhoud in ADP in vergelyking met ATP in alle transgeniese lyne en word verder ondersteun deur die ooruitdrukking van ADK, hoofsaaklik in die rigting van ADP produksie. Onderdrukte UMP-sintase in transgeniese suikerriet lei tot verhogings in sukrose, stysel en uridienilaat. UDP-glukose, hexose-fosfate en uridienilaat inhoud korreleer positief met die verandering in sukrose inhoud. Transgeniese lyne het verhoogde sukrose-fosfaatsintase (SPS) aktiwiteit en lae SuSy aktiwiteit wat dui op ‘n verandering in koolstof vloei in die rigting van sukrose. As gevolg van die afname in UMP-sintese aktiwiteit, word ‘n verhoogde reguleering van die bergingspad gesien, en dít hoofsaaklik via verhoogde aktiwiteit in uridienkinase (UK) wat positief korreleer met veranderinge in die uridienilaat poel. Addisioneel tot die onderdrukking van UMP-sintase was stysel inhoud en adenine- nucleotides in transgeniese lyne verhoog. Tabak plante wat getransformeer is met sitologiese UMP-sintase demonstreer verhoogde uitdrukking van die ensiem in al die transgeniese lyne. As ‘n resultaat van ooruitdrukking van UMP-sintase is sleutel metaboliete, onderandere sucrose, oorgereguleer. ‘n Verhoging in sukrose inhoud korreleer positief met beide hexose en hexose-fosfate maar nie met die uridienilaat poel nie. SPS aktiwiteit korreleer positief met die verhoging in sukrose inhoud en verklaar die meeste van die sukrose vervaardig in transgeniese lyne. Ten spyte van die verhoging in die adenilaat poel word geen noemenswaardige veranderinge gesien in die stysel inhoud nie. Die uitputtingsvlak van die UDP-glukose in alle transgeniese lyne was slegs ‘n aanduiding van die hoër aktiwiteit van UDP-glukose pirofosforilase (UGPase) in die formasie van glukose-1-fosfaat. Verder was geen bergingspad opgereguleer in die transgeniese lyne nie.
The South African Sugarcane Research Institute and the Gabonese Government who provided the financial support for this work
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Simpson, Luci N. "Cassette Systems for Creating Intergeneric Hybrid ATCases." Thesis, University of North Texas, 1999. https://digital.library.unt.edu/ark:/67531/metadc2237/.
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Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.
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Shen, Weiping. "Regulation of Escherichia coli pyrBI Gene Expression in Pseudomonas fluorescens." Thesis, University of North Texas, 1995. https://digital.library.unt.edu/ark:/67531/metadc278188/.
Full textAbstract:
Pseudomonas fluorescens does not appear to regulate the enzymes of de novo
pyrimidine biosynthesis at the level of gene expression. Little or no apparent repression of pyr gene expression is observed upon addition of exogenous pyrimidines to the growth medium. The Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) were sized down and cloned into the broad host range plasmid, pKT230. Upon introduction into a P.fluorescenspyrB mutant strain, ATCase showed repression in response to exogenously fed pyrimidine compounds. Thus, it was possible to bring about changes in pyrimidine nucleotide pool levels and in transcriptional regulation of gene expression at the same time.
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Azad, Kamran Nikkhah. "Pyrimidine Enzyme Specific Activity at Four Different Phases of Growth in Minimal and Rich Media, and Concomitant Virulence Factors Evaluation in Pseudomonas aeruginosa." Thesis, University of North Texas, 2005. https://digital.library.unt.edu/ark:/67531/metadc4918/.
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Pseudomonas aeruginosa is a Gram-negative rod, aerobic, non-fermenting, oxidase positive, pigment producing, and nutritionally versatile bacterium. Infections by P. aeruginosa are the most important cause of morbidity and mortality in immunocompromised patients, given virulence factor production that suppresses antibiotic therapy and promotes persistent infection. This research is the first comprehensive report of the pyrimidine biosynthetic pathway for all phases of growth in minimal and rich media coupled with the evaluation of virulence factor production of P. aeruginosa in comparison to four other bacterial species (Pseudomonas putida, Pseudomonas fluorescens, Burkholderia cepacia, and Escherichia coli wild-type strains). Cellular growth and passing genetic information to the next generation depend on the synthesis of purines and pyrimidines, the precursors of DNA and RNA. The pyrimidine biosynthetic pathway is essential and found in most organisms, with the exception of a few parasites that depend upon the pyrimidine salvage pathway for growth. Both the pyrimidine biosynthetic and salvage enzymes are targets for chemotherapeutic agents. In our laboratory, research on pyrimidine auxotrophic mutants showed the role of the pyrimidine biosynthetic pathway and its intermediates on P. aeruginosa metabolism and impaired virulence factors production. The present research shows that pyrimidine enzymes are active in all phases of growth, including the production of two forms of ATCase in the late log phase in P. aeruginosa. This finding may be explained by the displacement of the inactive PyrC' by the active PyrC or PyrC2 to form a new and larger pyrBC encoded ATCase. Pseudomonas aeruginosa wild-type appears to produce by far the most virulence factors, haemolysin, iron chelation, rhamnolipid, adherence, and three types of motility (swimming, swarming, and twitching) investigated in this study, when compared to the other four wild-type strains. Growth analysis was carried out as typically done in minimal medium but also in rich medium to simulate conditions in the blood and lung tissues of humans as P. aeruginosa infections develop.
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Hooshdaran, Sahar. "Characterization of Moraxella bovis Aspartate Transcarbamoylase." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc3012/.
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Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in the pyrimidine biosynthetic pathway. Bacterial ATCases have been divided into three classes, class A, B, and C, based on their molecular weight, holoenzyme architecture, and enzyme kinetics. Moraxella bovis is a fastidious organism, the etiologic agent of infectious bovine keratoconjunctivitis (IBK). The M. bovis ATCase was purified and characterized for the first time. It is a class A enzyme with a molecular mass of 480 to 520 kDa. It has a pH optimum of 9.5 and is stable at high temperatures. The ATCase holoenzyme is inhibited by CTP > ATP > UTP. The Km for aspartate is 1.8 mM and the Vmax 1.04 µmol per min, where the Km for carbamoylphosphate is 1.05 mM and the Vmax 1.74 µmol per min.
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XI, XU GUANG. "Les enzymes impliques dans la voie du metabolisme des pyrimidines : 1) etude des mecanismes moleculaires impliques dans la cooperativite et l'allosterie de l'aspartate transcarbamylase d'e. coli; 2) analyse du metabolisme pyrimidique lors du developpement des pneumoconioses." Paris 11, 1991. http://www.theses.fr/1991PA112033.
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Cette these se compose de deux parties distinctes. Une premiere partie de ce travail concerne, l'etude par mutagenese dirigee, des mecanismes moleculaires impliques dans la cooperativite et l'allosterie de l'aspartate transcarbamylase d'e. Coli. Dans un premier temps nous avons essaye de preciser le role de l'his134 dans la fixation des substrats et dans le mecanisme catalytique par des etudes de ph-dependance d'un mutant dans lequel l'his134 est remplace par une asparagine. L'implication de ce residu dans la fixation du groupement carbonyl du carbamylphosphate a ainsi pu etre etablie. De plus, il semble que ce residu ne soit pas implique dans un mecanisme de catalyse acide-base pour le transfert du carbamyl du carbamylphosphate. Par la suite, apres avoir effectue une serie de mutations portant sur la partie c-terminale de la sous-unite regulatrice, nous avons montre que l'interaction entre la partie c-terminale de la sous-unite regulatrice et la boucle 240 de la sous-unite catalytique est capitale pour le maintien de l'enzyme en etat t. Nous avons egalement mis en evidence le role crucial que joue cette partie de la molecule dans la transmission du signal du ctp. Enfin, nous avons reussi a identifier un residu dans la chaine regulatrice dont le role serait de discriminer le signal activation de celui d'inhibition. En effet, le remplacement de ce residu tyr77 par une phe conduit a une inversion des effets d'activation en inhibition. La deuxieme partie de ce travail concerne l'etude de la proliferation cellulaire observee lors de certains types de pneumoconioses. Nous avons analyse d'une part les effets de particules toxiques sur le metabolisme des ribonucleotides in vitro sur une lignee etablie de macrophages et d'autre part in vivo sur les macrophages alveolaires de rat. Il semblerait que les activites des enzymes impliques dans le metabolisme des pyrimidines ne soient pas affectees. Nous avons entrepri