The enzyme 3-deoxy-D-arabino-heptulusonate 7-phosphate synthase (DHS) catalyzes the first step of the shikimate pathway. In bacteria, the regulation of the pathway is mediated by allosteric inhibition of DHS by the aromatic amino acids tyrosine, phenylalanine and tryptophan. The regulation of the pathway in plants remains elusive but the aromatic amino acids are involved as suggested by the hypersensitivity of dhs1 knockout mutant to tyrosine. In this study the effects of the dhs1 mutation on endogenous levels of aromatic amino acids and of downstream metabolites are explored. HPLC analysis is used to measure levels of tyrosine and phenylalanine and 5-methyltryptophan sensitivity is used to probe levels of tryptophan. Additionally, the auxin content of whole seedlings was quantified by LC/MS and its local levels at the root apex are visualized with the DR5::GUS reporter system.

The enzyme 3-deoxy-D-arabino-heptulusonate 7-phosphate synthase (DHS) catalyzes the first step of the shikimate pathway. In bacteria, the regulation of the pathway is mediated by allosteric inhibition of DHS by the aromatic amino acids tyrosine, phenylalanine and tryptophan. The regulation of the pathway in plants remains elusive but the aromatic amino acids are involved as suggested by the hypersensitivity of dhs1 knockout mutant to tyrosine. In this study the effects of the dhs1 mutation on endogenous levels of aromatic amino acids and of downstream metabolites are explored. HPLC analysis is used to measure levels of tyrosine and phenylalanine and 5-methyltryptophan sensitivity is used to probe levels of tryptophan. Additionally, the auxin content of whole seedlings was quantified by LC/MS and its local levels at the root apex are visualized with the DR5::GUS reporter system.

E. coli Citrate Synthase (CS) is a large hexameric protein with a molecular weight of 280kDa and belonging to the type II class of citrate synthases. The crystal structure of E. coli CS in the T (inactive) state is known. The structure shows that the backbone atoms of 42 residues (1-5, 266-297 and 330-335) have temperature factors about 9 times greater than the average as compared to the rest of the molecule. Residues 266-297, also referred to as the “Mobile loop”, are particularly of interest since they form a part of the active site and any rearrangement in the mobile loop can provide useful information about the R(active) state of the protein. In this study, Nuclear Magnetic Resonance (NMR) Spectroscopy has been used to study the flexible regions of E. coli CS in solution. The flexible residues have been assigned based on the amino acid type by 15N-specific amino acid labeling, while the residue type has been assigned by site-directed mutagenesis. Changes in the dynamics of the flexible residues, in response to the substrate binding, have been studied using both NMR and Fluorescence Spectroscopy. Also a method to use Mass Spectrometry for accessing the isotopic incorporation in the samples prepared for NMR spectroscopy has been described. The initial hypothesis in this study was that only the mobile loop residues which show significant high B-factors will contribute to the NMR spectrum. However in the NMR spectrum, in addition to the mobile loop, some uncharacterized flexible regions were also observed. We also found that some of the residues show signs of slow conformational exchange resulting in multiple signals in the NMR spectrum. In addition we see that the environment of some flexible residues is changed in the presence of substrates, a few residues were immobilized, but most remained mobile. / February 2007

Thèse d'exercice : Pharmacie : Université de Nantes : 2003. / Bibliogr. f. 170-182 [120 réf.].

Background: Significant interest in homocysteine exists due to its established role in embryogenesis, cardiovascular disease and neurotoxicity. This research investigates total plasma homocysteine in the context of carcinogenesis among healthy individuals aged 20 to 50 years. It is hypothesized that during this timeframe, elevated total plasma homocysteine (tHcy) concentration implicates a breakdown of the methionine-homocyteine biosynthesis pathway, in which folate deficiency, oxidative stress and altered DNA methylation capacity are potential consequences relevant to cancer etiology. Purpose: The overall purpose of this research is to identify novel genetic predispositions to a biomarker of cancer risk (tHcy). Interactions with dietary and genetic factors that act on this pathway are explored. Methods: The study population consisted of 284 healthy male and female volunteers recruited in Kingston, Ontario and Halifax, Nova Scotia between 2006 and 2008. Specifically, polymorphisms under consideration included: i) the thymidylate synthase enhancer region (TSER) tandem repeat polymorphism and ii) the GC single nucleotide polymorphism (G/C SNP) both found on the 5’untranslated region (UTR) of the TS gene, and iii) the 6 base pair deletion at base pair 1494 (TS1494del6) found on the 3’UTR. TS polymorphisms were categorized based on either 5’ or 3’ location and were dichotomized to either high or low TS expression. Gene-gene interactions between polymorphisms in TS and methylenetetrahydrofolate reductase (MTHFR C677T) on tHcy concentration were also analyzed. In addition, gene-diet interactions between serum folate and vitamin B12 status were examined. Results: Mean tHcy concentration for this study population was 8.65 µmol/L (standard deviation=1.96 µmol/L). After adjustment for confounders, higher mean tHcy levels of 0.48 μmol/L and 0.46 μmol/L were observed for the main effects of 5’polymorphisms (5’High) (p=0.04) and 3’polymorphism (3’High) (p=0.05), respectively. The largest difference in mean tHcy concentration was observed for the joint effects of TS polymorphisms (µ=0.74 μmol/L, p=0.11). Gene-gene interaction was observed between TS and MTHFR polymorphisms on tHcy concentrations (p<0.01). Conclusions: The findings of this research provide evidence of an association between TS polymorphisms and tHcy concentrations. These results suggest that TS polymorphisms, independent of dietary factors, may lead to elevated tHcy levels and potentially contribute to cancer development. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2008-08-21 16:05:02.797

No description available.

N-Acetylneuraminic acid synthase (NANAS) is an enzyme responsible for the biosynthesis of N-acetylneuraminic acid (NANA). NANA is the most common form of a group of nine carbon sugar molecules called the sialic acids. NANA production is common in mammalian cells for vital physiological processes. A few species of microorganisms, including pathogenic bacteria such as Neisseria meningitidis and Campylobacter jejuni, are known to synthesise NANA by their bacterial NANAS. These pathogenic bacteria synthesise NANA for molecular mimicry, allowing them to evade the host immune system. This thesis examines the NANAS enzymes from N. meningitidis and C. jejuni. Inhibitory studies with these enzymes were explored by performing enzyme kinetics with substrate analogues and a product analogue which structurally mimic the natural substrates or product of NANAS. Inhibition constants were determined for a variety of analogues to give insight in to how the enzyme accommodates its substrates within the active site of NANAS. This study may be a useful step in the development of alternative antibiotics for bacterial meningitis and other diseases in the future. NANAS catalyses a condensation reaction between phosphoenolpyruvate (PEP) and N-acetyl mannosamine (ManNAc). Both PEP and ManNAc analogues were explored as inhibitors of the enzymes. Results from this study show that increasing steric bulk of the substituents at C3 of PEP unexpectedly delivers more potent inhibition of the enzyme. This finding suggests that a slightly modified binding position of the PEP analogue within the PEP binding site of the enzyme may be responsible for the inhibition. A reduced acyclic analogue of ManNAc was found to be an effective inhibitor of the enzymes. This finding indicates how important the acyclic form of ManNAc is in the reaction mechanism catalysed by this enzyme.

E. coli Citrate Synthase (CS) is a large hexameric protein with a molecular weight of 280kDa and belonging to the type II class of citrate synthases. The crystal structure of E. coli CS in the T (inactive) state is known. The structure shows that the backbone atoms of 42 residues (1-5, 266-297 and 330-335) have temperature factors about 9 times greater than the average as compared to the rest of the molecule. Residues 266-297, also referred to as the “Mobile loop”, are particularly of interest since they form a part of the active site and any rearrangement in the mobile loop can provide useful information about the R(active) state of the protein. In this study, Nuclear Magnetic Resonance (NMR) Spectroscopy has been used to study the flexible regions of E. coli CS in solution. The flexible residues have been assigned based on the amino acid type by 15N-specific amino acid labeling, while the residue type has been assigned by site-directed mutagenesis. Changes in the dynamics of the flexible residues, in response to the substrate binding, have been studied using both NMR and Fluorescence Spectroscopy. Also a method to use Mass Spectrometry for accessing the isotopic incorporation in the samples prepared for NMR spectroscopy has been described. The initial hypothesis in this study was that only the mobile loop residues which show significant high B-factors will contribute to the NMR spectrum. However in the NMR spectrum, in addition to the mobile loop, some uncharacterized flexible regions were also observed. We also found that some of the residues show signs of slow conformational exchange resulting in multiple signals in the NMR spectrum. In addition we see that the environment of some flexible residues is changed in the presence of substrates, a few residues were immobilized, but most remained mobile.

No description available.

No description available.