Dissertations / Theses on the topic 'Amino acids – Biosynthesis'
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Waluk, Dominik Paweł. "Biosynthesis and physiological functions of N-acyl amino acids." Doctoral thesis, Stockholms universitet, Institutionen för genetik, mikrobiologi och toxikologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-75766.
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N-acyl amino acids are lipid signalling molecules that have recently been identified in biological systems. These lipids are structurally related to the endocannabinoids, although they do not activate cannabinoid receptors. In 2001, N-arachidonoyl glycine was the first signalling lipid in this group to be identified in bovine and rat brain and since then, about 50 novel N-acyl amino acids have been identified in mammalian systems. These N-acyl amino acids are involved in regulating pain processes, are anti-inflammatory and regulate body temperature, but the metabolic pathways for production and metabolism remain poorly understood. This thesis focussed on the identification of pathways for production and regulation of N-acyl amino acids, in particular N-acyl glycines, and in identifying physiological functions for N-acyl amino acids (particularly N-acyl taurines). Our results identified an enzymatic pathway for production of N-acyl glycines in human and we identified that the human glycine N-acyltransferase-like 2 (hGLYATL2) conjugates (amidates) medium- and long-chain, saturated and unsaturated acyl-CoAs with glycine, to produce N-acyl glycines, with the preferential production of N-oleoyl glycine. Furthermore, we have characterized two other members of the gene family of glycine N-acyltransferases (GLYATs) in human, the hGLYATL1 and hGLYATL3 that may be involved in the production of N-acyl amino acids. As N-acyl glycines are bioactive signalling molecules, it is likely their production requires a rapid on/off switch. The post-translational modification of proteins can result in enzyme regulation, without the need for transcriptional regulation. We have identified that hGLYATL2 is regulated by acetylation/deacetylation on lysine 19, and using mutation analysis, we show that deacetylation of lysine 19 is important for full enzyme activity. The physiological functions of N-acyl amino acids are not well studied to date. In this thesis, we have identified that N-arachidonoyl taurine and N-oleoyl taurine trigger insulin secretion by increasing the calcium flux in pancreatic b-cells via the activation of transient receptor potential vanilloid subfamily 1 (TRPV1). This work on N-acyl amino acids has led us to identify new pathways and physiological functions for these lipid signalling molecules, which advances our knowledge of the importance of these lipids in mammalian systems.At the time of doctoral defence the following papers were unpublished and had a status as follows:Paper 2: Accepted; Paper 3: Manuscript; Paper 4; Manuscript
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Krappmann, Sven Kurt. "Biosynthesis of Aromatic Amino Acids in Yeast and Aspergillus." Doctoral thesis, [S.l. : s.n.], 2000. http://hdl.handle.net/11858/00-1735-0000-000D-F20C-1.
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Barnard, Sandra H. "Amalgamation of Nucleosides and Amino Acids in Antibiotic Biosynthesis." UKnowledge, 2013. http://uknowledge.uky.edu/pharmacy_etds/20.
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The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading frame which encodes a putative serine hydroxymethyltransferase (SHMT). The revelation of this shared open reading frame resulted in the proposal that this putative SHMT catalyzes an aldol-type condensation reaction utilizing glycine and uridine-5’-aldehyde, resulting in the GlyU core. The enzyme LipK involved in A-90289 biosynthesis was used as a model to functionally assign this putative SHMT to reveal its functions as an l-threonine: uridine-5’-aldehyde transaldolases. Biochemical analysis indicates enzymatic activity is dependent upon pyridoxal-5’-phosphate, is non-reactive with alternative amino acids, and produces acetaldehyde as a co-product. Structural characterization of the enzymatic product is consistent with (5’S,6’S)-GlyU indicating that this enzyme orchestrates a C-C bond breaking and formation resulting in two new stereocenters to make a new l-a-amino acid. The same activity was demonstrated for the LipK homologues involved in the biosynthesis of muraminomicin, A-503083, and A-102395. This l-threonine: uridine-5’-aldehyde transaldolase was used with alternative aldehyde substrates to prepare unusual l-a-amino acids, suggesting the potential for exploiting this enzyme to make new compounds.
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Brown, Judy Forsyth. "Regulation in aromatic amino acid biosynthesis in Saccharomyces cerevisiae." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/12947.
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Murphey, Roberta Jean. "Synthesis of deoxyhypusine in eukaryotic initiation factor 4D in rat hepatoma cells." Diss., The University of Arizona, 1989. http://hdl.handle.net/10150/184691.
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The aim of this research was to study the mechanism involved in the synthesis of deoxyhypusine, the intermediate step in the synthesis of the amino acid hypusine. Oeoxyhypusine is derived from the butylamine moiety of a spermidine molecule which is added to the famino group of one lysine in the eukaryotic initiation factor 40 (eIF-4D). Initially, a hepatoma tissue cell (HTC) lysate with a pH of 9.5 in glycine buffer and with a depleted spermidine pool supported deoxyhypusine synthesis in protein. Since CHES buffer was as efficient as glycine buffer, the synthesis of deoxyhypusine was pH dependent (optimum ∼9.2) and not buffer dependent. Next, several inhibitors were used in the cell-free system to block deoxyhypusine synthesis. Only guazatine, a plant amine oxidase inhibitor, completely inhibited deoxyhypusine synthesis. This suggested that an oxidase was involved in deoxyhypusine synthesis. In addition factors were investigated as possible allosteric stimulators of deoxyhypusine formation. NAD⁺, NADH, FAD⁺, FMN⁺, and as nicotinamide were tested for effects on deoxyhypusine formation. NAD⁺ was the most efficient stimulator, but NAOH and nicotinamide also stimulated deoxyhypusine formation. Although these factors increased the synthesis of deoxyhypusine, these assays were done in buffer with low concentrations of spermidine. When the spermidine pool was replenished, these effects were diminished. Thus, it appeared that NAD⁺ may lower the apparent K(m) for spermidine without affecting the V(max) of deoxyhypusine synthesis. The inhibition of deoxyhypusine synthesis by guazatine implied the involvement of a polyamine oxidase. Therefore, the effect of oxygen depletion on deoxyhypusine formation was investigated. The depletion of oxygen reduced the level of deoxyhypusine synthesis to 12% of the control. This activity could be restored to 85% by reoxygenation of the lysate. Thus in support of the suggestion made by the guazatine data, a spermidine oxidase in involved in deoxyhypusine formation. The most significant contribution of this work was the development of a cell free system to study deoxyhypusine. This synthesis required an unusually high pH in vitro and required polyamine depletion (Chapter 2). In addition, synthesis requires a unique spermidine oxidase that is blocked by a guazatine and is conditionally stimulated by NAD⁺ (Chapter 3).
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Ng, S. C. "Enzymatic and non enzymatic synthesis of amino acid derivatives." Thesis, University of Oxford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379948.
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Nili, Nafisseh. "Limitations to amino acid biosynthesis de novo in ruminal strains of Prevotella and Butyrivibrio." Title page, contents and abstract only, 1996. http://web4.library.adelaide.edu.au/theses/09PH/09phn712.pdf.
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Bibliography: leaves 226-261. Investigates nitrogen utilization in some species of rumen bacteria with the object of understanding the role of ammonia versus exogenous amino acids in relation to microbial growth.
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Lee, Johnny Chien-Yi Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Transcriptional and metabolic responses of yeast Saccharomyces cerevisiae to the addition of L-serine." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/41012.
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Sudden changes in nutrient resources are common in the natural environment. Cells are able to adapt and propagate under changing environmental conditions by making adjustments in their cellular processes. These cellular adaptations involve genome-wide transcriptional reprogramming that results in the induction or repression of metabolic pathways. Specific enzymes are then synthesised and activated to maximise the use of the newly available nutrient sources. L-serine is one of the twenty proteinogenic amino acids, and can be synthesised in yeast by the glycolytic and gluconeogenic pathways when growing on fermentable or non-fermentable carbon sources or taken up from the environment when available. L-serine is metabolically linked to glycine and is a predominant donor of one-carbon units in one-carbon metabolism. L-serine is also a source of pyruvate and ammonia and contributes to other cellular processes including the biosynthesis of cysteine and phospholipids. Previous work has shown that yeast cells exhibit transcriptional induction of the one-carbon pathway and the genes involved in the synthesis of purine and methionine after the addition of 10 mM glycine. Here it is shown that addition of 10 mM L-serine did not, however, elicit the same transcriptional response. This is primarily due to differences in the uptake of glycine and L-serine in yeast. High concentrations of extracellular L-serine were required for yeast to show an increase in intracellular L-serine concentration of the magnitude required to trigger a noticeable cellular response. Despite L-serine and glycine being interconvertable via the SHMT isozymes and being a one-carbon donor, the genome-wide transcriptional response exhibited by cells in response to L-serine addition was markedly different to that seen for glycine. The predominant response to an increase in intracellular L-serine was the induction of the general amino acid control system and the CHA1 gene encoding the serine (threonine) dehydratase. Unlike glycine, addition of L-serine triggered only minor induction of the one-carbon pathway. A large portion of intracellular L-serine was converted to pyruvate and ammonia in the mitochondrion as the result of induction of CHA1. The high intracellular concentration of L-serine stimulated the cell to increase the production of oxaloacetate and to increase the biosynthesis of L-aspartate. Transient increases in the intracellular L-glutamate and L-glutamine were also observed after the addition of L-serine. The work presented in this study shows that large increase in the intracellular concentration of amino acid is required to trigger a significant transcriptional response. Yeast cells exhibit different transcriptional and metabolic responses to the addition of L-serine and glycine even though these two amino acids are closely metabolically linked. Addition of L-serine provokes the GAAC response, expression of the CHA1 gene and stimulates the biosynthesis of L-aspartate in yeast whereas addition of glycine induces the one-carbon pathway which leads to the biosynthesis of the purine nucleotides.
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Chan, K. K. Jason. "Identification and enzyme studies of rare amino acid biosynthesis from Streptomyces cattleya." Thesis, University of St Andrews, 2013. http://hdl.handle.net/10023/4478.
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This thesis is focussed on the biosynthesis of three toxins: fluoroacetate, 4-fluoro-L-theronine and β-ethynyl-L-serine which are biosynthesised by the soil bacteria Streptomyces cattleya. The two fluorinated metabolites originate from a common biosynthetic pathway and the thesis describes studies carried out on an aldose-ketose isomerase enzyme of the pathway. The biosynthetic origin of β-ethynyl-L-serine is not known. A total synthesis of this acetylenic amino acid is descibed along with the development of a new analytical method for identifying the metabolite and for future isotope-labelling based biosynthetic studies. Chapter 1 presents the background of this research. It is focussed on the biosynthesis of fluoroacetate and 4-fluoro-L-threonine by S. cattleya and it also introduces alkyne-containing natural products and their biosynthesis. Chapter 2 describes the work carried out on crystallisation of the aldose-ketose isomerase of the fluorometabolite pathway in S. cattleya. Crystals of the isomerase were obtained and they were diffracted by X-ray, however a structure could not be solved. Chapter 3 contains site-directed mutagenesis studies of the isomerase from S. cattleya. Chapter 4 describes an enantioselective total synthesis of β-ethynyl-L-serine. A robust analytical technique based on derivatisation using 'Click' chemistry and LC-MS was developed for the detection of this amino acid directly from the fermentation broth. Chapter 5 details the experimental procedures for compounds synthesised in this thesis and the biological procedures for gene cloning and protein purification.
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Al-Bar, Omar Abdulrahman Mostafa. "Modified amino acids and peptides as potential inhibitors of bacterial cell wall biosynthesis." Thesis, University of Southampton, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303364.
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Widboom, Paul Fredrick. "Biosynthesis and Incorporation of Nonproteinogenic Amino Acids into Non-ribosomal Peptide Natural Products." Thesis, Boston College, 2008. http://hdl.handle.net/2345/1366.
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Thesis advisor: Steven D. BrunerComplex and unique enzymology is often behind the biosynthesis of natural products. This thesis is focused on how non-proteinogenic amino acids are biosynthesized and then incorporated into natural products. Chapters two, three and four deal with a unique dioxygenase found in vancomycin biosynthesis. Chapter five elaborates on the biochemical characterization along with efforts toward structural characterization of a terminal non-ribosomal peptide synthetase module. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. We have solved the first structure of an enzyme of this oxygenase class complexed with a bound substrate mimic. The use of a designed, synthetic substrate analog allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation
Thesis (PhD) — Boston College, 2008
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Choroba, Oliver Werner. "Enzymes involved in crosslinking and biosynthesis of the rare amino acids in glycopeptide antibiotics." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621185.
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Wang, C. (Chunguang). "Human lysyl hydroxylase isoforms:multifunctionality of human LH3 and the amino acids important for its collagen glycosyltransferase activities." Doctoral thesis, University of Oulu, 2002. http://urn.fi/urn:isbn:9514267990.
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Abstract
Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen.
Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types.
Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E.coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E.coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product.
The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.
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Christianson, Carl Victor. "Understanding The Biosynthesis And Utilization Of Non-Proteinogenic Amino Acids For The Production Of Secondary Metabolites In Bacteria." Thesis, Boston College, 2008. http://hdl.handle.net/2345/967.
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Thesis advisor: Steven D. BrunerBacteria utilize complex enzymatic machinery to create diverse secondary metabolites. The architectural complexities of these small molecules are enhanced by nature’s ability to synthesize non-proteinogenic amino acids for incorporation into these scaffolds. Many of these natural products are utilized as therapeutic agents, and it would be advantageous to understand how the bacteria create various non-natural amino acid building blocks. With a greater understanding of these systems, engineering could be used to create libraries of potentially useful natural product analogs. The tyrosine aminomutase SgTAM from the soil bacteria Streptomyces globisporus catalyzes the formation of tyrosine to generate (S)-B-tyrosine. The precise mechanistic role of MIO in this novel family of aminomutases has not been established. We report the first X-ray crystal--> structure of an MIO based aminomutase and confirm the structural homology of SgTAM to ammonia lyases. Further work with mechanistic inhibitors provide structural evidence of the mechanism by which MIO dependent enzymes operate. We have also investigated LnmQ, an adenylation domain in the biosynthetic pathway of leinamycin. Leinamycin is an antitumor antibiotic that was isolated from soil samples in 1989. LnmQ is responsible for the specific recognition of D-alanine and subsequent activation as an aminoacyl adenylate species. We have cloned the gene into a DNA vector and expressed it in E. coli. Upon purification of the protein, crystallization conditions have been tested. Synthesis of an inhibitor that mimics the aminoacyl adenylate product catalyzed by LnmQ has been completed. Crystallization with this--> inhibitor will provide better quality crystals and a catalytically informative co-complex
Thesis (PhD) — Boston College, 2008
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Clingman, Carina C. "A Feedback Loop Couples Musashi-1 Activity to Omega-9 Fatty Acid Biosynthesis: A Dissertation." eScholarship@UMMS, 2014. http://escholarship.umassmed.edu/gsbs_diss/718.
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All living creatures change their gene expression program in response to nutrient availability and metabolic demands. Nutrients and metabolites can directly control transcription and activate second-‐messenger systems. In bacteria, metabolites also affect post-‐transcriptional regulatory mechanisms, but there are only a few isolated examples of this regulation in eukaryotes. Here, I present evidence that RNA-‐binding by the stem cell translation regulator Musashi-‐1 (MSI1) is allosterically inhibited by 18-‐22 carbon ω-‐9 monounsaturated fatty acids. The fatty acid binds to the N-‐terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. I identify stearoyl-‐CoA desaturase-‐1 as a MSI1 target, revealing a feedback loop between ω-‐9 fatty acid biosynthesis and MSI1 activity. To my knowledge, this is the first example of an RNA-‐binding protein directly regulated by fatty acid. This finding may represent one of the first examples of a potentially broad network connecting metabolism with post-‐transcriptional regulation.
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Kräbs, Gudrun. "Wavelength dependent induction and biosynthesis of UV-absorbing mycosporine-like amino acids in marine macroalgae = Wellenlängen-abhängige Induktion und Biosynthese von UV-absorbierenden Mykosporine-ähnlichen Aminosäuren in marinen Makroalgen /." Bremerhaven : Alfred-Wegener-Inst. für Polar- und Meeresforschung, 2004. http://www.gbv.de/dms/bs/toc/389858625.pdf.
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Maderazo, Alan Baer. "A Study on the Cellular Localization of Factors Involved in Yeast Nonsense-Mediated mRNA Decay and their Mechanisms of Control on Nonsense mRNA Translation: a Dissertation." eScholarship@UMMS, 2000. https://escholarship.umassmed.edu/gsbs_diss/105.
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Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance mechanism conserved in eukaryotes. This thesis explores several interesting aspects of the NMD pathway. One important aspect of NMD which is presently the subject of intense controversy is the subcellular localization of NMD. In one set of experiments, the decay kinetics of the ade2-1 and pgk1 nonsense mRNAs (substrates for NMD) were investigated in response to activating the NMD pathway to determine if cytoplasmic nonsense mRNAs are immune to NMD in the yeast system. The results of these studies demonstrated that activation of NMD caused rapid and immediate degradation of both the ade2-1 and the early nonsense pgk1 steady state mRNA populations. The half lives of the steady state mRNA populations for both ade2-1 and pgk1 (early nonsense) were shortened from >30 minutes to approximately 7 minutes. This was not observed for pgk1mRNAs that contained a late nonsense codon demonstrating that activation of NMD specifically targeted the proper substrates in these experiments. Therefore, in yeast, nonsense mRNAs residing in the cytoplasm are susceptible to NMD. While these findings are consistent with NMD occurring in the cytoplasm, they do not completely rule out the possibility of a nuclear-associated decay mechanism.
To investigate the involvement of the nucleus in NMD, the putative nuclear targeting sequence identified in Nmd2p (one of the trans-acting factors essential for NMD) was characterized. Subcellular fractionation experiments demonstrated that the majority of Nmd2p localized to the cytoplasm with a small proportion detected in the nucleus. Specific mutations in the putative nuclear localization signal (NLS) of Nmd2p were found to have adverse effects on the protein's decay function. These effects on decay function, however, could not be attributed to a failure in nuclear localization. Therefore, the residues that comprise the putative NLS of Nmd2p are important for decay function but do not appear to be required for targeting the protein to the nucleus. These results are in accordance with the findings above which implicate the cytoplasm as an important cellular compartment for NMD.
This thesis then investigates the regulatory roles of the trans-acting factors involved in NMD (Upf1p, Nmd2p, and Upf3p) using a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100). Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression. Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy can1-100 expression in wild-type cells caused mRNA stabilization similar to that obtained in upf/nmd cells but did not result in comparable suppression. can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA. These findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay.
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Ghosh, Shubhendu. "Cross-Talk Between Factors Involved in mRNA Translation and Decay: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/454.
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The proper workings of an organism rely on the accurate expression of genes throughout its lifetime. An important determinant for protein production is the availability of template mRNA molecules, the net effect of which is governed by their rates of synthesis vs. their rates of degradation. Normal mRNAs are proposed to be relatively stable in the cytoplasm while present in a protective, circularized conformation – the closed loop – through eIF4G-bridged interactions with 3’-bound poly(A) binding protein (Pab1p) and 5’-bound eIF4E. Introduction of a premature nonsense codon into an otherwise normal mRNA results in its rapid destabilization in cells, suggesting that not all stop codons behave the same, and events at premature termination events that lead to accelerated degradation of nonsense-containing mRNAs likely differ from those at normal termination, in which normal decay rates are maintained. The enhanced degradation observed for nonsense-containing mRNAs occurs through an evolutionarily conserved pathway involving the products of the UPF1, UPF2/NMD2, and UPF3 genes, the precise biochemical roles of which have remained elusive. We have developed a yeast cell-free translation system that allows us to assay biochemical events occurring at premature termination codons, compare them to those occurring at normal terminators, and study the role of Upf1p in these events. We find that premature termination is an inefficient process compared to normal termination and that one outcome of termination at a premature termination codon (PTC) is reinitiation at a nearby start codon. This in vitro post-termination reinitiation phenotype is dependent on the presence of Upf1p, a finding we have recapitulated in vivo. We also developed biochemical assays to define a role for Upf1p in translation following premature termination in vitro and find that Upf1p is involved in post-termination ribosome dissociation and reutilization. Supporting this idea are our findings that Upf1p predominantly cosediments with purified 40S ribosomal subunits. Finally, using our in vitro translation/toeprinting system, we have further characterized events leading to the formation of the mRNA closed loop structure and find that two states of the closed loop exist. The first requires the preinitiation 48S complex and includes Pab1p, eIF4G, eIF4E, and eIF3, whereas the second is formed after 60S joining and additionally requires the translation termination factors eRF1 and eRF3.
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Linscott, Kristin Brooke. "DISCOVERING A NOVEL ANTIFUNGAL TARGET IN DOWNSTREAM STEROL BIOSYNTHESIS USING A SQUALENE SYNTHASE FUNCTIONAL MOTIF." UKnowledge, 2017. http://uknowledge.uky.edu/biochem_etds/33.
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The sterol biosynthetic pathway is essential for growth of all eukaryotic cells and the main target of antifungal agents. The emergence of resistance to these antifungals in an already ill patient population indicates a need to develop drugs that have a broad spectrum of activity among pathogenic fungi and have minimal patient toxicity. Squalene synthase is the first committed step in the sterol pathway and has been studied intensively for development of antifungal agents. While the overall architecture of this enzyme is identical throughout eukaryotes, it was shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implies that there is a component of the fungal carboxy-terminal domain that is responsible for the complementation phenotype and that is unique to the fungal kingdom of life.
To determine the role of the carboxy-terminal domain of squalene synthase in the sterol pathway, we used the yeast Saccharomyces cerevisiae with a squalene synthase knockout mutation and expressed squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. When induced, non-fungal squalene synthases could not complement the knockout mutation and instead led to the accumulation of carboxysterol intermediates, suggesting an interaction between squalene synthase and the downstream sterol C4-decarboxylase. Overexpression of a sterol C4-decarboxylase from any kingdom of life both decreased the accumulation of carboxysterol intermediates and allowed non-fungal squalene synthases to complement the squalene synthase knockout mutation.
Using chimeric squalene synthases from each kingdom of life, the motif in the C-terminal domain responsible for preventing this toxicity was mapped to a kingdom-specific 26-amino acid hinge motif adjacent to the catalytic domain. Furthermore, over-expression of the carboxy-terminal domain alone containing a hinge motif from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Since this hinge region is unique to and highly conserved within each kingdom of life, this data provides evidence for the development of an antifungal therapeutic as well as for tools to develop an understanding of triterpene catalytic activity and identify similar motifs in other biosynthetic pathways.
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Jiang, Ying. "Transfer of the Ribosome-Nascent Chain Complex to the Translocon in Cotranslational Translocation: A Thesis." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/332.
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Cotranslational translocation is initiated by targeting of a ribosome-bound nascent polypeptide chain (RNC) to the endoplasmic reticulum (ER) membrane. The targeting reaction is coordinated by the signal recognition particle (SRP) through its interaction with the RNC and the membrane-bound SRP receptor (SR). A vacant translocon is a prerequisite for the subsequent nascent chain release from SRP-SR-RNC complex. It has been proposed that the protease-accessible cytosolic domains of the Sec61p complex play an important role in posttargeting steps by providing the binding site for the ribosome or interacting with the SR to initiate the signal sequence releasing. In this study, we have investigated the detailed mechanism that allows transfer of the ribosome-nascent chain (RNC) from the SRP-SR complex to the translocon using yeast S. cerevisiaeas the model system.
Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in translocation of nascent polypeptides that utilize the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome binding activity, indicating that the L6 and L8 sec61mutants impact different steps in the cotranslational translocation pathway.
An interaction between the signal recognition particle receptor (SR) and the Sec61 complex has been proposed to facilitate transfer of the ribosome-nascent chain (RNC) complex to an unoccupied translocon. The slow growth and cotranslational translocation defects caused by deletion of the transmembrane span of yeast SRβ (srp102pΔTMD) are exaggerated upon disruption of the SSH1 gene, which encodes the pore subunit of a cotranslational translocation channel. Disruption of the SBH2 gene, which encodes the β-subunit of the Ssh1p complex, likewise causes a synthetic growth defect when combined with srp102pΔTMD. The in vivo kinetics of translocon gating by RNCs were slow and inefficient in the ssh1Δ srp102pΔTMD mutant. A critical role for translocon β-subunits in SR recognition is supported by the observation that deletion of both translocon β-subunits causes a block in the cotranslational targeting pathway that resembles elimination of either subunit of the SR, and could be partially suppressed by expression of carboxy-terminal Sbh2p fragments.
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Busch, Silke. "Amino Acid Biosynthesis and the COP9 Signalosome in Aspergillus nidulans." Doctoral thesis, [S.l.] : [s.n.], 2002. http://hdl.handle.net/11858/00-1735-0000-0006-AE62-6.
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Krashes, Michael Jonathan. "Systems Level Processing of Memory in the Fly Brain: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/419.
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Understanding the mechanisms of memory is vital in making sense of the continuity of the self, our experience of time and of the relation between mind and body. The invertebrate Drosophila melanogaster offers us an opportunity to study and comprehend the overwhelming complexity of memory on a smaller scale. The work presented here investigates the neural circuitry in the fly brain required for olfactory memory processing. Our observation that Dorsal Paired Medial (DPM) neurons, which project only to mushroom body (MB) neurons, are required during memory storage but not for acquisition or retrieval, led us to revisit the role of MB neurons in memory processing. We show that neurotransmission from the α'β' subset of MB neurons is required to acquire and stabilize aversive and appetitive odor memory but is dispensable during memory retrieval. In contrast neurotransmission from MB αβ neurons is only required for memory retrieval. These data suggest a dynamic requirement for the different subsets of MB neurons in memory and are consistent with the notion that recurrent activity in a MB α'β' neuron-DPM neuron loop is required to consolidate memories formed in the MB αβ neurons. Furthermore, we show that a single two-minute training session pairing odor with an ethologically relevant sugar reinforcement forms long-term appetitive memory that lasts for days. This robust, stable LTM is protein-synthesis-, Creb- and radish-dependent and relies on the activity in the DPM neuron and mushroom body α'β' neuron circuit during the first hour after training and mushroom body αβ neuron output during retrieval. Lastly, experiments feeding and/or starving flies after training reveals a critical motivational drive that enables memory retrieval. Neural correlates of motivational states are poorly understood, but using our assay we found a neural mechanism that accounts for this motivation-state-dependence. We demonstrate a role for the Neuropeptide F (dNPF) circuitry, which led to the identification of six dopaminergic MB-MP neurons that innervate the mushroom bodies as being critical for appetitive memory performance. Directly blocking the MB-MP neurons releases memory performance in fed flies whereas stimulating them suppresses memory performance in hungry flies. These studies provide us with an enhanced knowledge of systems level memory processing in Drosophila.
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Banerji, Suneale. "Aromatic amino acid biosynthesis in Pneumocystis carinii." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336091.
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Gaston, Marsha. "Biosynthesis of pyrrolysine, the 22nd amino acid." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1318533705.
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Longstaff, David Gordon. "Requirements and rationale for amber translation as pyrrolysine." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1196107921.
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Sun, Lele. "Peptidyltransfer Reaction Catalyzed by the Ribosome and the Ribozyme: a Dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/115.
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The "RNA world" hypothesis makes two predictions that RNA should have been able both to catalyze RNA replication and to direct protein synthesis. The evolution of RNA-catalyzed protein synthesis should be critical in the transition from the RNA world to the modem biological systems. Peptide bond formation is a fundamental step in modem protein biosynthesis. Although many evidence suggests that the ribosome is a ribozyme, peptide bond formation has not been achieved with ribosomal RNAs only. The goal of this thesis is to investigate whether RNA could catalyze peptide bond formation and how RNA catalyzes peptide bond formation. Two systems have been employed to approach these questions, the ribozyme system and the ribosome system. Ribozymes have been isolated by in vitro selection that can catalyze peptide bond formation using the aminoacyl-adenylate as the substrate. The isolation of such peptide-synthesizing ribozymes suggests that RNA of antiquity might have directed protein synthesis and bolsters the "RNA world" hypothesis. In the other approach, a novel assay has been established to probe the ribosomal peptidyltransferase reaction in the presence of intact ribosome, ribosomal subunit, or ribosomal RNA alone. Several aspects of the peptidyltransfer reaction have been examined in both systems including metal ion requirement, pH dependence and substrate specificity. The coherence between the two systems is discussed and their potential applications are explored. Although the ribozyme system might not be a reminiscence of the ribosome catalysis, it is still unique in other studies. The newly established assay for ribosomal peptidyltransferase reaction provides a good system to investigate the mechanism of ribosomal reaction and may have potential application in drug screening to search for the specific peptidyltransferase inhibitors.
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Kan, Ming-Chung. "Analysis of CPEB Family Protein Member CPEB4 Function in Mammalian Neurons: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/362.
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Local protein synthesis is required for long-term memory formation in the brain. One protein family, Cytoplasmic Polyadenylation Element binding Protein (CPEB) that regulates protein synthesis is found to be important for long-term memory formation possibly through regulating local protein synthesis in neurons. The well-studied member of this family, CPEB1, mediates both translational repression and activation of its target mRNAs by regulating mRNA polyadenylation. Mouse with CPEB1 KO shows defect in memory extinction but not long-term memory formation. Three more CPEB1 homologs (CPEB2-4) are identified in mammalian system. To test if CPEB2-4 may have redundant role in replacing CPEB1 in mediating local protein synthesis, the RNA binding specificity of these homologs are studied by SELEX. The result shows CPEB2-4 bind to RNAs with consensus sequence that is distinct from CPE, the binding site of CPEB1. This distinction RNA binding specificity between CPEB1 and CPEB2-4 suggests CPEB2-4 cannot replace CPEB1 in mediating local protein synthesis. For CPEB2-4 have distinct RNA binding specificity compared to CPEB1, they are referred as CPEB-like proteins. One of CPEB-like protein, CPEB3, binds GluR2 mRNA and represses its translation. The subcellular localization of CPEB family proteins during glutamate over stimulation is also studied. The CPEB family proteins are identified as nucleus/cytoplasm shuttling proteins that depend on CRM1 for nuclear export. CPEB-like proteins share similar nuclear export ciselement that is not present in CPEB1. Over-stimulation of neuron by glutamate induces the nuclear accumulation of CPEB family proteins possibly through disrupted nuclear export. This nuclear accumulation of CPEB family protein is induced by imbalance of calcium metabolism in the neurons. Biochemical and cytological results suggest CPEB4 protein is associated with ER membrane peripherally in RNA independent manner. This research provides general description of biochemical, cytological properties of CPEB family proteins.
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Davis, Danny Thomas. "The stereoselective synthesis of amino acids for biosynthetic studies." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq24771.pdf.
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McKernan, Philip Andrew. "Manipulation of the shikimate pathway in Streptomyces coelicolor." Thesis, University of Glasgow, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296048.
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Campbell, Samantha Alison. "The second enzyme of histidine biosynthesis from Arabidopsis thaliana." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325254.
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Campbell, Sara Jane. "Amino acid biosynthesis pathways as anti-microbial targets in Acanthamoeba." Thesis, University of the West of Scotland, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.544489.
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Against the background of growing competition in the global marketplace, understanding customers, is a significant aspect of marketing. In the search for competitive advantage, there is a need to measure service quality to better understand its antecedentsa nd consequencesa, nd establish methods for its improvement. In the Libyan economy, the banking sector is one of the most important. Its significance increased after the 2003 lifting of the United Nations sanction. This was followed by entry to the sector of a number of domestic and multinational firms. Despite this increased competition, domestic banks are still widely considered to suffer from low levels of service quality. The main purpose of this study is to evaluate the actual level of service quality provided by Libyan public commercial banks as perceived by their customers. A modified SERVQUAL model was developed to measure service quality in Libyan commercial public banks. The resulting instrument is intended to help these banks to measure their service quality and focus on the service quality dimensions of most importance to their customers. It also aimed to gain an understanding of cultural and environmental influences on service quality in the Libyan banking sector, and their effect on banking management practices. It is also expected that this instrument, and its results, will contribute to future research into service quality. The findings of the present study have produced some important results. Firstly, the level of service quality offered by the Libyan public commercial banks as it was perceived by their customers was relatively high. Secondly, the theoretical five-factor structure of the SERVQUAL model was not confirmed in the Libyan banking context, and the service quality structure in the Libyan context appears to be four-dimensional. Furthermore, the study offers suggestions to banking managers to allocate their resources more efficiently to the most important dimensions, i. e. reliability and tangibles, to improve service quality, since the factor analysis indicates that these are the most important dimensions to customers. Finally, reflections on the methods used to modify SERVQUAL to make it more sensitive to a particular cultural context have implications for future researchersin terms of methodology, method and data analysisbiosynthesis pathway is present and essential for Acanthamoeba growth. We have cloned and sequenced the sixth enzyme in this pathway, imidazole glycerol-phosphate dehydratase (IGPD) (EC 4.2.1.19), from A. castellanii. This enzyme is the molecular target for 3-amino-1, 2, 4-triazole (3AT), a commonly used commercially available herbicide. The growth of A. castellanii was restricted by 3AT and this inhibition was ablated in a dose dependent manner by the addition of histidine to the medium. 3AT was also not toxic to RCE cells. This demonstrates that 3AT acts specifically to inhibit IGPD and that the histidine biosynthesis pathway is present in A. castellanii. This study provides the first evidence of the histidine biosynthesis pathway in a protozoan and demonstrates its potential as a target for therapeutic intervention during Acanthamoeba infection.
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Hachisuka, Shinichi. "Studies on coenzyme and amino acid biosynthesis in hyperthermophilic archaea." Kyoto University, 2018. http://hdl.handle.net/2433/232489.
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Stuart, Fiona. "An investigation of aromatic amino acid biosynthesis in Streptomyces rimosus." Thesis, University of Glasgow, 1990. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506454.
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Gulko, Miriam Kolog. "A non-canonical pathway for aromatic amino acid biosynthesis in haloarchea." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-131276.
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Llewellyn, Nicholas Michael. "Biosynthesis of butirosin : incorporation of the unique (S)-4-amino-2-hydroxybutyryl amino acid side chain." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612089.
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Larue, Ross C. "A novel aminoacyl-tRNA synthetase and its amino acid, pyrrolysine, the 22nd genetically encoded amino acid." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1261432150.
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Batson, Sarah. "Structural enzymology of peptidoglycan biosynthetic D-amino acid dipeptide ligases." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/3117/.
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This thesis describes approaches to further understand the structure and enzymology of D-Ala-D-Ala ligases (DDL) and those ligases with altered second substrate specificity, which confer glycopeptide antibiotic resistance. DDL is an essential enzyme in the biosynthetic pathway of the bacterial cell wall peptidoglycan. The approaches described are based primarily on the previous transition state mimic; VanA and EcDdlB, co-crystal structures. Active site mapping of VanA by site directed mutagenesis yielded VanA mutants that were expressed and purified for kinetic studies. The active site mapping of VanA supports the predictions about catalysis from the X-ray structure of VanA, but failed to identify a catalytic base. Additionally, the first enzymatic characterisation of a VanD ligase was performed. This analysis revealed that VanD4 ligase is a less efficient D-Ala-D-Lac ligase, and selected for a wider variety of second substrates in comparison to VanA. EcDdlB was expressed in E.coli, purified and crystallised. Three structures of EcDdlB have been solved to 1.4-1.7Å resolutions, representing a product inhibition complex, a ternary complex, and a D-cycloserine inhibited complex. Based upon the latter EcDdlB structure, we propose a novel suicide-substrate mechanism for the Dcycloserine mediated inhibition of DDLs, involving phosphorylation of D-cycloserine. Future studies will validate this mechanism. Finally, contributions towards rational design of EcDdlB and VanA inhibitors have been made. This work has significantly increased knowledge in the field of DDL enzymes, progressing towards the exploitation of these targets for antibiotic development. The deposition of PDB files of three new EcDdlB structures will provide the tools for further rational based drug design campaigns against EcDdlB. The novel mechanism of Dcycloserine inhibition may also have implications for the further development of inhibitors of DDLs.
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Clarke, Tyler Brooke. "Studies on the inhibitor selectivity and inhibitory signal transfer of a-Isopropylmalate synthase." Thesis, University of Canterbury. Chemistry, 2013. http://hdl.handle.net/10092/11303.
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α-Isopropylmalate synthase (α-IPMS) is responsible for catalysing the first committed step in leucine biosynthesis. This pathway is found in plants and microorganisms, including pathogenic bacteria such as Mycobacterium tuberculosis and Neisseria meningitidis. α-IPMS catalyses a Claisen condensation reaction between α-ketoisovalerate (KIV) and acetyl coenzyme A (AcCoA) to form the product α-isopropylmalate (IPM). This enzyme undergoes feedback inhibition by the end product of the pathway, leucine. This regulation allows the control of the rate leucine biosynthesis.
This project focuses on the α-IPMS enzymes from M. tuberculosis and N. meningitidis (MtuIPMS and NmeIPMS). These α-IPMS enzymes are homodimeric in structure. Each monomer consists of a catalytic domain which comprises of a (β/α)8 barrel fold, two subdomains and a regulatory domain, to which the allosteric binding of the natural inhibitor leucine occurs. The mechanism by which the allosteric binding of leucine leads to a decrease in enzymatic activity is not yet fully understood.
Citramalate synthase (CMS) is responsible for catalysing the first committed step of threonine-independent isoleucine biosynthesis. This enzyme is extremely similar to α-IPMS in both the reaction which it catalyses and the catalytic and regulatory domain structure. CMS catalyses a Claisen condensation reaction between pyruvate and AcCoA to produce citramalate (CM). CMS is also feedback inhibited by the end product of its pathway, isoleucine.
The similarity between α-IPMS and CMS enzymes resulted in and examination of the inhibitor selectivity of MtuIPMS. Amino acids in the leucine binding site were altered to their counterparts in the isoleucine binding site of the CMS enzyme to see if the selectivity of the leucine binding site could be interchanged.
Results from this study show that it is possible to change inhibitor selectivity with a single amino acid substitution. However, changing the selectivity from leucine to isoleucine was unsuccessful. Instead, one of the MtuIPMS variants displayed significantly increased sensitivity to an alternative amino acid, norvaline. The MtuIPMS variants were expressed and purified using immobilised metal affinity chromatography and size-exclusion chromatography. These variants were then kinetically characterised and displayed similar binding affinities and turnover rates for the natural substrates to the wild-type enzyme. As expected changes to the leucine binding pocket had drastic effects on the sensitivity of the enzyme to its natural inhibitor. This work is described in Chapter 2 of this thesis.
The mechanism by which the regulatory signal is transferred from the allosteric leucine binding site to the catalytic site in α-IPMS is not fully understood. NmeIPMS variants were created based on preliminary molecular dynamic simulations which indicated that significant changes in residue contacts were associated with leucine binding. Chapter 3 describes studies that explore the effect of single amino acid substitutions of NmeIPMS. The NmeIPMS variants were expressed and purified similarly to MtuIPMS, using immobilised metal affinity chromatography and size-exclusion chromatography. Variants were subsequently characterised via mass spectrometry, differential scanning fluorimetry and kinetic assays. It was found that each variant generated retained sensitivity to leucine but displayed significant differences in the catalytic efficiencies with AcCoA. One of the generated variants also displayed a significant increase in thermal stability.
Results are drawn together in Chapter 4 along with future directions of this research. This chapter details knowledge gained into protein structure and allosteric mechanisms in this thesis.
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Rice, Christopher Aaron. "Histidine and Lysine amino acid biosyntheis as antimicrobial targets in Acanthamoeba species." Thesis, University of the West of Scotland, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627899.
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Acanthamoeba species have been found to be facultative, opportunistic, protozoan, parasites. They are found ubiquitously in the environment. Acanthamoeba species are emerging pathogens causing a severe corneal infection called Acanthamoeba keratitis (AK) in immunocompetent individuals normally associated with contact lens wearers, which may result in blindness. Many drugs used to treat Acanthamoeba infections are poorly effective because they can induce encystation and/or fail to eliminate cysts, so more reliable and effective therapies are required. To this end, the studies described herein demonstrate the ability of Acanthamoeba unlike its mammalian host to synthesise histidine and lysine. Thus growth of Acanthamoeba spp. was restricted by 3-amino-1, 2, 4-triazole (3AT) (IC50 125μM), an inhibitor of imidazoleglycerol-phosphate dehydratase (IGPD) (EC 4.2.1.19) a key enzyme for de novo histidine biosynthesis. The addition of histidine was able to ablate the effects of 3AT in a dose dependent manner when added to cultures, indicating that 3AT was specifically acting on histidine biosynthesis. 3AT had minimal cytotoxicity to a prostate cancer cell line (PC3-luc) at concentrations that restrict the growth of Acanthamoeba. Bioinformatic analysis demonstrated that Acanthamoeba has the full complement of enzymes necessary for histidine biosynthesis. The complete coding sequence for a novel multifunctional gene, histidinol dehydrogenase (EC 1.1.1.23) and IGPD (EC 4.2.1.19) have both been amplified from A. castellanii and A. polyphaga cDNA, both have been cloned and sequenced. The gene encoding histidinol dehydrogenase from Acanthamoeba has been found to encode several enzymes including imidazoleglycerol phosphate synthase, phosphoribosyl-formimino-5-amino-1-imidazole-carboxyamide ribotide isomerase, phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphatase, histidinol dehydrogenase and ATP Phosphoribosyltransferase. An experimental compound (C348) previously demonstrated to inhibit IGPD of plant species was found to restrict the growth of Acanthamoeba (IC50 305nM). This inhibition was also ablated with the addition of exogenous histidine when added to the medium. C348 showed no cytotoxicity effects to the PC3-luc at concentrations restricting the growth of Acanthamoeba species.
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Todd, Catherine. "Investigations into 2,3-dihydroxy acid intermediates on the branched-chain amino acid biosynthetic pathway." Thesis, University of Warwick, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308022.
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Yanamadala, Srinivasa Rao. "Molecular cloning and characterization of regulatory enzymes in threonine biosynthetic pathway from soybean." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4932.
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Thesis (M.S.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on May 12, 2009) Includes bibliographical references.
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Robinson, N. G. "New amino acid syntheses : Applications to studies on penicillin biosynthesis and C-nucleoside synthesis." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375322.
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Li, Yanyan. "Biosynthesis of butirosin : elucidation of the pathway to (2S)-4-amino-2-hydroxybutyric acid." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614989.
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Canu, Nicolas. "New insights into the recognition of the substrates of cyclodipeptide synthases." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS580.
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Les cyclodipeptides constituent, avec leurs dérivés plus complexes les dicétopiperazines (DKP), une importante famille de produits naturels, synthétisés essentiellement par des micro-organismes. Une approche intéressante pour synthétiser une grande diversité de DKP consiste à étudier et manipuler les voies de biosynthèse de ces molécules. Les synthases de cyclodipeptides (CDPS) constituent une famille d’enzymes dédiés à la production de cyclodipeptides, qui ont la particularité d’utiliser les ARNt aminoacylés (AA-ARNt) comme substrats. Afin d’exploiter complètement le potentiel de ces enzymes, il est nécessaire de mieux comprendre leur spécificité, notamment vis-à-vis de substrats non naturels. Dans cette thèse, nous avons tout d’abord démontré que les CDPS sont capables d’incorporer des acides aminés non naturels, en utilisant la promiscuité des aminoacyl-ARNt synthétases (AARS) d’Escherichia coli. Puis nous avons amélioré notre compréhension de la reconnaissance de la partie ARNt des susbtrats par les CDPS. En utilisant les flexizymes, des ribozymes à activité AARS, nous avons généré des analogues d’AA-ARNt avec des parties ARNt tronquées. Nous avons pu montrer que des « mini AA-ARNt » reproduisant les 7 paires de bases du bras accepteur des ARNt sont d’aussi bons substrats que les AA-ARNt complets, ce qui suggère que les CDPS interagissent principalement avec les bras accepteurs de leurs substrats ARNtCyclodipeptides and their complex derivatives, the diketopiperazines (DKPs), constitute a large class of natural products with diverse and noteworthy pharmacological activities observed for many naturally occurring DKPs. A promising approach to generate diverse DKPs is to study and manipulate DKP biosynthetic pathways. Cyclodipeptide synthases (CDPSs) constitute an enzyme family dedicated to the synthesis of cyclodipeptides, with the particularity to use aminoacylated-tRNAs (AA-tRNAs) as substrates. In order to unlock the biosynthetic potential of these enzymes, better understanding their specificity, in particular towards non-natural substrates, is required.In this thesis, we first significantly expanded the diversity of cyclodipeptides accessible with CDPSs by showing that CDPSs could incorporate non-canonical amino acids, through the use of the promiscuity of E. coli aminoacyl-tRNA synthetases. Then, we gave new insights into the recognition by CDPSs of the tRNA moieties of their substrates. By using an innovative RNA acylation strategy based on a class of ribozymes called flexizymes, we generated analogues of AA-tRNAs with truncated RNA moieties. Among these “AA-minitRNAs”, we showed that those mimicking the entire 7 bp stems of tRNAs are as good substrates as AA-tRNAs, which suggests that CDPSs interact mainly with the acceptor arms of tRNAs and paves the way for promising biophysical and structural studies
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Follettie, Maximillian Todd. "DNA technology for Corynebacterium glutamicum : isolation and characterization of amino acid biosynthetic genes." Thesis, Massachusetts Institute of Technology, 1989. http://hdl.handle.net/1721.1/16501.
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Henderson, Douglas Brian. "The Development of a New Cloning Strategy for the Biosynthetic Production of Brush-Forming Poly(Amino Acids)." Diss., Virginia Tech, 2004. http://hdl.handle.net/10919/30103.
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The design and discovery of new surface-active polymers that self-assemble on solid substrates to form brush layers will have a major impact on numerous applications. Through recombinant DNA technology, there exists the potential to harness a cell's protein synthesis machinery to produce a brush-forming poly(amino acid) (or PAA) with an exactly specified amino acid sequence, thus controlling the polymer's composition at a level unequaled by conventional organic polymer synthesis. The presented work demonstrates the cloning, expression, purification and characterization of de novo-designed PAA's designed to form brush layers on alumina surfaces. Using conventional recombinant DNA methods, the feasibility of producing a PAA consisting of a poly-glutamate block and a poly-proline block was demonstrated. However, the PAA design was limited by the inherent limitations of conventional cloning techniques.
We introduce here the development of a simple and versatile strategy for producing de novo-designed, high molecular weight PAA's using recombinant DNA technology. The basis of this strategy is that small DNA modules encoding for short PAA blocks can be easily inserted directly into a commercially available and unmodified expression vector. The insertions can be made repeatedly until the gene encodes for a polymer of desired molecular weight and composition. Thus, sequential modifications can be made to the PAA without having to re-start the gene assembly process from the beginning, thereby allowing for quick determination of how these changes affect polymer structure and function. The feasibility and simplicity of this method was shown during the production of a PAA, consisting of a long zwitterionic tail block and a short acidic anchor block, designed to form optimal brush layers on alumina surfaces. The success and flexibility of this method indicates that it can be applied for production of de novo-designed polypeptides in general. It is hoped that this method will contribute towards the rapid development of bio-inspired protein-based polymers for a variety of applications.
This dissertation also contains research that aimed to use phage display technology to develop a new liposome-based immunoassay against biological toxins. This work was part of a collaboration effort with the U.S. Department of Defense and Luna Innovations.Ph. D.
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Barton, Michael D. "Evolutionary Systems Biology of Amino Acid Biosynthetic Cost and Gene Importance in Saccharomyces Cerevisiae." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518546.
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Buabeng, Emmnauel Ramsey. "Towards the Synthesis of Novel Glycomimetics of N-Acetyl-2-amino-2-deoxy-D-mannopyranose uronic acid (D-ManNAcA) and Derivatives." Youngstown State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1471478050.
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Dahal, Gopal Prasad. "Development of Selective Inhibitors against Enzymes Involved in the Aspartate Biosynthetic Pathway for Antifungal Drug Development." University of Toledo / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532889045486984.
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Tran, David. "Investigating the substrate specificity of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase." Thesis, University of Canterbury. Chemistry, 2011. http://hdl.handle.net/10092/6565.
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The shikimate pathway is a biosynthetic pathway that is responsible for producing a variety of organic compounds that are necessary for life in plants and microorganisms. The pathway consists of seven enzyme catalysed reactions beginning with the condensation reaction between D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) to give the seven-carbon sugar DAH7P. This thesis describes the design, synthesis and evaluation of a range of alternative non-natural four-carbon analogues of E4P (2- and 3-deoxyE4P, 3-methylE4P, phosphonate analogues of E4P) to probe the substrate specificity of different types of DAH7P synthases [such as Mycobacterium tuberculosis (a type II DAH7PS), Escherichia coli (a type Ialpha DAH7PS) and Pyrococcus furiosus (a type Ibeta DAH7PS)].