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1
蘇雅頌 and Ngar-chung Nellie So. "Pyrimidine nucleotide biosynthesis in adult angiostrongylus Cantonensis (Nematoda : Metastrongyloidea)." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1993. http://hub.hku.hk/bib/B3123320X.
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So, Ngar-chung Nellie. "Pyrimidine nucleotide biosynthesis in adult angiostrongylus Cantonensis (Nematoda : Metastrongyloidea) /." [Hong Kong : University of Hong Kong], 1993. http://sunzi.lib.hku.hk/hkuto/record.jsp?B13637745.
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Guo, Wenyue. "A study of structure and function of two enzymes in pyrimidine biosynthesis." Thesis, Boston College, 2012. http://hdl.handle.net/2345/2772.
Full textAbstract:
Thesis advisor: Evan R. KantrowitzNucleotides, the building blocks for nucleic acids, are essential for cell growth and replication. In E. coli the enzyme responsible for the regulation of pyrimidine nucleotide biosynthesis is aspartate transcarbamoylase (ATCase), which catalyzes the committed step in this pathway. ATCase is allosterically inhibited by CTP and UTP in the presence of CTP, the end products of the pyrimidine pathway. ATP, the end product of the purine biosynthetic pathway, acts as an allosteric activator. ATCase undergoes the allosteric transition from the low-activity and low-affinity T state to the high-activity and high-affinity R state upon the binding of the substrates. In this work we were able to trap an intermediate ATCase along the path of the allosteric transition between the T and R states. Both the X-ray crystallography and small-angle X-ray scattering in solution clearly demonstrated that the mutant ATCase (K164E/E239K) exists in an intermediate quaternary structure shifted about one-third toward the canonical R structure from the T structure. The structure of this intermediate ATCase is helping to understand the mechanism of the allosteric transition on a molecular basis. In this work we also discovered that a metal ion, such as Mg2+, was required for the synergistic inhibition by UTP in the presence of CTP. Therefore, the metal ion also had significant influence on how other nucleotides effect the enzyme. A more physiological relevant model was proposed involving the metal ion. To better understand the allosteric transition of ATCase, time-resolved small-angle X-ray scattering was utilized to track the conformational changes of the quaternary structure of the enzyme upon reaction with the natural substrates, PALA and nucleotide effectors. The transition rate was increased with an increasing concentration of the natural substrates but became over one order of magnitude slower with addition of PALA. Addition of ATP to the substrates increased the rate of the transition whereas CTP or the combination of CTP and UTP exhibited the opposite effect. In this work we also studied E. coli dihydroorotase (DHOase), which catalyzes the following step of ATCase in the pyrimidine biosynthetic pathway. A virtual high throughput screening system was employed to screen for inhibitors of DHOase, which may become potential anti-proliferation and anti-malarial drug candidates. Upon the discovery of the different conformations of the 100's loop of DHOase when substrate or product bound at the active site, we've genetically incorporated an unnatural fluorescent amino acid to a site on this loop in the hope of obtaining a better understanding of the catalysis that may involve the movement of the 100's loop
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
4
Harris, Katharine Morse. "Studies of structure, function and mechanism in pyrimidine nucleotide biosynthesis." Thesis, Boston College, 2012. http://hdl.handle.net/2345/2594.
Full textAbstract:
Thesis advisor: Evan R. KantrowitzThesis advisor: Mary F. Roberts
Living organisms depend on enzymes for the synthesis using small molecule precursors of cellular building blocks. For example, the amino acid aspartate is synthesized in one step by the amination of oxaloacetate, an intermediate compound produced in the citric acid cycle, exclusively by means of an aminotransferase enzyme. Therefore, function of this aminotransferase is critical to produce the amino acid. In the Kantrowitz Lab, we seek to understand the molecular rational for the function of enzymes that control rates for the biosynthesis of cellular building blocks. If one imagines the above aspartate-synthesis example as a single running conveyer belt, any oxaloacetate that finds its way onto that belt will be chemically transformed to give aspartate. We can extend this notion of a conveyer belt to any enzyme. Therefore, the rate at which the belt moves dictates the rate of synthesis. Now imagine many, many conveyer belts lined in a row to give analogy to a biosynthesis pathway requiring more than one enzyme for complete chemical synthesis. This is such the case for the biosynthesis of nucleotides and glucose. Nature has developed clever tricks to exquisitely control the rate of product output but means of altering the rate of one or some of the belts in the line of many, without affecting the rate of others. This type of biosynthetic rate regulation is termed allostery. Studies described in this dissertation will address questions of allosteric processes and the chemistry performed by two entirely different enzymes and biosynthetic pathways. The first enzyme of interest is fructose-1,6-bisphosphatase (FBPase) and its role in the biosynthesis of glucose. Following FBPase introduction in Chapter One, Chapter Two describes the minimal atomic scaffold necessary in a new class of allosteric type 2 diabetes drug molecules to effect catalytic inhibition of Homo sapiens FBPase. Following, is the second enzyme of interest, aspartate transcarbamoylase (ATCase) and its role in the biosynthesis of pyrimidine nucleotides. Succeeding ATCase introduction in Chapter Three, Chapter Four describes a body of work exclusively about the catalysis by ATCase. This work was inspired by the human form of the enzyme following the human genome project completion providing data that show likely Homo sapiens ATCase is not allosterically regulated. Chapter Five describes work on a allosterically-regulated, mutant ATCase and provides a biochemical model for the molecular rational for the catalytic inhibition upon cytidine triphosphate (CTP) binding to the allosteric site. The experimental techniques used for answering research questions were enzyme X-ray crystallography, in silico docking, kinetic assay experiments, genetic sub-cloning and genetic mutation
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
5
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.
6
Cockrell, Gregory Mercer. "New Insights into Catalysis and Regulation of the Allosteric Enzyme Aspartate Transcarbamoylase." Thesis, Boston College, 2013. http://hdl.handle.net/2345/3156.
Full textAbstract:
Thesis advisor: Evan R. KantrowitzThe enzyme aspartate transcarbamoylase (ATCase) is an enzyme in the pyrimidine nucleotide biosynthetic pathway. It was once an attractive target for anti-proliferation drugs but has since become a teaching model due to kinetic properties such as cooperativity and allostery exhibited by the Escherichia coli form of the enzyme. ATCase from E. coli has been extensively studied over that last 60 years and is the textbook example of allosteric enzymes. Through this past research it is understood that ATCase is allosterically inhibited by CTP, the end product of pyrimidine biosynthesis, and allosterically activated by ATP, the end product of the parallel purine biosynthetic pathway. Part of the work discussed in this dissertation involves further understanding the catalytic properties of ATCase by examining an unregulated trimeric form from Bacillus subtilis, a bacterial ATCase that more closely resembles the mammalian form than E. coli ATCase. Through X-ray crystallography and molecular modeling, the complete catalytic cycle of B. subtilis ATCase was visualized, which provided new insights into the manifestation of properties such as cooperativity and allostery in forms of ATCase that are regulated. Most of the work described in the following chapters involves understanding allostery in E. coli ATCase. The work here progressively builds a new model of allostery through new X-ray structures of ATCase*NTP complexes. Throughout these studies it has been determined that the allosteric site is bigger than previously thought and that metal ions play a significant role in the kinetic response of the enzyme to nucleotide effectors. This work proves that what is known about ATCase regulation is inaccurate and that currently accepted, and taught, models of allostery are wrong. This new model of allostery for E. coli ATCase unifies all old and current data for ATCase regulation, and has clarified many previously unexplainable results
Thesis (PhD) — Boston College, 2013
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
7
Montigny, Jacky de. "Ura5 et ura10, deux genes codant pour deux isoenzymes a activite omp pyrophosphorylase chez la levure saccharomyces cerevisiae : structure, expression et regulation." Strasbourg 1, 1988. http://www.theses.fr/1988STR13198.
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Lortet, Sylviane. "Les mécanismes de synthèse des nucléotides pyrimidiques myocardiques à partir de la cytidine." Grenoble 1, 1987. http://www.theses.fr/1987GRE10056.
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La synthese des nucleotides pyrimidiques du tissu myocardique a ete mesuree sur une preparation de coeur isole et perfuse. Les constantes cinetiques sont determinees a partir des extraits acellulaires, une forte activite kinasique est notee vis a vis de l'uridine et de la cytidine. L'ensemble des resultats montre que la phosphorylation de la cytidine plasmatique pourrait representer la voie essentielle de synthese des nucleotides pyrimidiques myocardiques
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.
10
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/.
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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.
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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.
12
Entezampour, Mohammad. "Characterization of pyrimidine biosynthesis in Acinetobacter calcoaceticus using wild type and mutant strains." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc798038/.
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Pyrimidine nucleotide biosynthesis was studies in Acinetobacter calcoaceticus ADP-1. Pyrimidine auxotrophic mutants were isolated and characterized for this purpose. One such Pyr mutant, strain ADP-1-218 was chosen for further study.
13
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/.
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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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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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McGaughey, Kathleen M. "The effects of protein associations on pyrimidine deoxyribonucleotide biosynthesis." Thesis, 2001. http://hdl.handle.net/1957/29400.
Full textAbstract:
The faithful replication of DNA depends on the appropriate balance of DNA
precursors. From studies conducted in bacteriophage T4, models for
deoxyribonucleotide biosynthesis producing pools appropriate for DNA replication
have made it possible to understand more complex systems. A portion of that body
of evidence supports the concept that deoxyribonucleotide biosynthesis for
bacteriophage T4 is carried out by an association of enzymes and other cellular
components in a complex called the dNTP synthetase complex. This dissertation
explores potential direct protein-protein interactions within this complex for the
preparation of pyrimidine deoxyribonucleotides.
Direct associations for enzymes involved in pyrimidine deoxyribonucleotide
biosynthesis were examined by affinity chromatography. It was determined that there
was a significant direct relationship between T4 thymidylate synthase and T4 dCMP
deaminase, between T4 dCTPase/dUTPase and T4 dCMP deaminase as well. The
interaction between thymidylate synthase and dCMP deaminase was significantly
influenced by the presence of dCTP, a positive effector of dCMP deaminase.
Furthermore, protein associations changed the kinetic character of pyrimidine
deoxyribonucleotide production. T4 dCTPase/dUTPase, a member of the dNTP
synthetase complex, significantly alters the kinetic nature of thymidylate synthase by
working with thymidylate synthase in a reciprocal relationship. T4 single-stranded
DNA binding protein, a member of the replication complex, alters the activity of
thymidylate synthase as well. Attempts to isolate a kinetically coupled complex from
two or more constituent proteins of the dNTP synthetase complex were frustrated by
protein degradation to fragments under 10 kDa in size.
Pyrimidine deoxyribonucleotide synthesis is located between the significant
energy investment of ribonucleotide reductase and phosphate attachments by kinases
to prepare the deoxyribonucleotide molecules for DNA replication. In bacteriophage
T4, intermediate reactions are driven by mass action but are modulated by subtleties
including direct protein associations and the presence of small molecules that
influence enzyme function. Through these and potentially similar controls, pools of
deoxyribonucleotides are prepared and delivered in a timely, balanced manner to the
DNA replication apparatus.Graduation date: 2002