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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
71

I. Understanding Membrane Interactions of Bacterial Exoproteins; II. Identification and Characterization of a Novel Mammalian cis-Aconitate Decarboxylase

Cheng, Jiongjia January 2013 (has links)
Thesis advisor: Mary F. Roberts / Secreted phosphatidylinositol-specific phospholipase Cs (PI-PLCs) are often virulence factors in pathogenic bacteria. Understanding how these enzymes interact with target membranes may provide novel methods to control bacterial infections. In this work, two typical PI-PLC enzymes, from Bacillus thuringiensis (Bt) and Staphylococcus aureus (Sa), were studied and their membrane binding properties were examined and correlated with enzymatic activity. BtPI-PLC is kinetically activated by allosteric binding of a phosphatidylcholine (PC) molecule. MD simulations of the protein in solution suggested correlated loop and helix motions around the active site could regulate BtPI-PLC activity. Vesicle binding and enzymatic studies of variants of two proline residues, Pro245 and Pro254, that were associated with these motions showed that loss of the correlated motions between the two halves of PI-PLC were more critical for enzymatic activity than for vesicle binding. Furthermore, loss of enzyme activity could be rescued to a large extent with PC present in a vesicle. This suggests that binding to PC changes the enzyme conformation to keep the active site accessible. SaPI-PLC shows 41.3% sequence similarity with BtPI-PLC but has very different ways its activity is regulated. While it is kinetically activated by PC it does not in fact bind to that phospholipid. Enzymatic and membrane interaction assays showed that SaPI-PLC has evolved a complex, apparently unique way to control its access to PI or GPI-anchored substrate. (i) An intramolecular cation-pi latch facilitates soluble product release under acidic conditions without dissociation from the membrane. (ii) There is a cationic pocket on the surface of enzyme that likely modulates the location of the protein. (iii) Dimerization of protein is enhanced in membranes containing phosphatidylcholine (PC), which acts not by specifically binding to the protein, but by reducing anionic lipid interactions with the cationic pocket that stabilizes monomeric protein. SaPI-PLC activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane depleted in anionic phospholipids. This protein also served as a way to test the hypothesis that a cation-pi box provides for PC recognition site. This structural motif was engineered into SaPI-PLC by forming N254Y/H258Y. This variant selectively binds PC-enriched vesicles and the enzyme binding behavior mimics that of BtPI-PLC. Itaconic acid (ITA) is a metabolite synthesized in macrophages and related cell lines by a cis-aconitate decarboxylase (cADC). cADC activity is dramatically increased upon macrophage stimulation. In this work, the cell line RAW264.7 was used to show that cADC activity upon stimulation requires de novo protein synthesis. MS analyses of partially purified RAW264.7 protein extracts from stimulated cells show a large increase for immunoresponsive gene 1 protein (IRG1) and siRNA knockdown of the IRG1 reduces cADC activity upon stimulation. Suspected active site residues of IRG1 were identified by mutagenesis studies of the recombinant protein based on a homology structure model of fungal cADC. The cloning and overexpression of this enzyme should help clarify the cofactor-independent decarboxylation mechanism of this mammalian enzyme as well as open up future studies into the specific role of ITA in the mammalian immune system and cancers. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
72

Subunit Interactions in the Inducible Arginine Decarboxylase from Escherichia Coli B

Depusoy, Catalina N. 01 May 1983 (has links)
The nature of the subunit interactions in the inducible arginine decarboxylase from Escherichia coli B is of considerable interest because of the observed differences in the catalytic activities of the dimer and the decamer; the decamer is active and the dimer is inactive. To study these interactions, inactive dimers were prepared by sodium borohydride reduction of the E-amino--pyridoxal-P Schiff base. Hybrid decamers were then prepared from varying molar ratios of native and reduced dimers. The hybrid decamers were indistinguishable from native decamers as observed in the analytical ultracentrifuge and on acrylamide gel electrophoresis. Kinetic studies indicated that true hybrids were formed rather than mixtures of all-native and all-reduced decamers. Results obtained with the decamers containing 1, 2, 3, or 4 parts in 5 of reduced enzyme showed no significant changes in Km values from the native decamer. However, the Vm values for these hybrids are greater than predicted from the mole fraction of active dimers. For example, the hybrid containing 20% reduced enzyme approaches the Vm of the native decamer. These observations suggest that, in the intact molecule, two active sites cooperate catalytically but only one is catalytically active.
73

Challenges in Enzyme Catalysis - Photosystem II and Orotidine Decarboxylase : A Density Functional Theory Treatment

Lundberg, Marcus January 2005 (has links)
<p>Possibly the most fascinating biochemical mechanism remaining to be solved is the formation of oxygen from water in photosystem II. This is a critical part of the photosynthetic reaction that makes solar energy accessible to living organisms.</p><p>The present thesis uses quantum chemistry, more specifically the density functional B3LYP, to investigate a mechanism where an oxyl radical bound to manganese is the active species in O-O bond formation. Benchmark calculations on manganese systems confirm that B3LYP can be expected to give accurate results. The effect of the self-interaction error is shown to be limited. Studies of synthetic manganese complexes support the idea of a radical mechanism. A manganese complex with an oxyl radical is active in oxygen formation while manganese-oxo complexes remain inactive. Formation of the O-O bond requires a spin transition but there should be no effect on the rate. Spin transitions are also required in many short-range electron-transfer reactions.</p><p>Investigations of the superproficient enzyme orotidine decarboxylase support a mechanism that involves an invariant network of charged amino acids, acting together with at least two mobile water molecules.</p>
74

Challenges in Enzyme Catalysis - Photosystem II and Orotidine Decarboxylase : A Density Functional Theory Treatment

Lundberg, Marcus January 2005 (has links)
Possibly the most fascinating biochemical mechanism remaining to be solved is the formation of oxygen from water in photosystem II. This is a critical part of the photosynthetic reaction that makes solar energy accessible to living organisms. The present thesis uses quantum chemistry, more specifically the density functional B3LYP, to investigate a mechanism where an oxyl radical bound to manganese is the active species in O-O bond formation. Benchmark calculations on manganese systems confirm that B3LYP can be expected to give accurate results. The effect of the self-interaction error is shown to be limited. Studies of synthetic manganese complexes support the idea of a radical mechanism. A manganese complex with an oxyl radical is active in oxygen formation while manganese-oxo complexes remain inactive. Formation of the O-O bond requires a spin transition but there should be no effect on the rate. Spin transitions are also required in many short-range electron-transfer reactions. Investigations of the superproficient enzyme orotidine decarboxylase support a mechanism that involves an invariant network of charged amino acids, acting together with at least two mobile water molecules.
75

Characterization of the activities of trans-3-chloroacrylic acid dehalogenase and cis-3-chloroacrylic acid dehalogenase and malonate semialdehyde decarboxylase homologues : mechanism and evolutionary implications

Serrano, Hector, doctor of pharmacy 05 September 2012 (has links)
Members of the tautomerase superfamily are characterized by a [beta-alpha-beta] structural fold motif as well as a catalytic N-terminal proline (Pro-1). Three members of the superfamily are involved in the degradation of the nematocide 1,3-dichloropopene; trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and malonate semialdehyde decarboxylase (MSAD). CaaD and cis-CaaD are involved in the hydration of their respective 3-chloroacrylic acid isomers to generate malonate semialdehyde. Subsequently, MSAD is responsible for catalyzing the decarboxylation of malonate semialdehyde to generate acetaldehyde. All three of these enzymes contain an N-terminal proline (Pro-1) that functions as a general acid, in contrast to other tautomerase superfamily members, such as 4-oxalocrotonate tautomerase (4-OT) and macrophage migration inhibitory factor (MIF), where Pro-1 acts as a catalytic base. Two new members of the tautomerase superfamily have been cloned and characterized; FG41 MSAD, a homologue of MSAD from Coryneform Bacterium strain FG41, and Cg10062, a homologue of cis-CaaD from Corynebacterium glutamicum, with low-level cis-CaaD and CaaD activities. As part of an effort to delineate the mechanisms of CaaD, cis-CaaD and Cg10062, secondary activities for all three enzymes were characterized. The three enzymes function as efficient phenylpyruvate tautomerases (PPT), converting phenylenolpyruvate to phenylpyruvate. The activity also indicates that the active site of these three enzymes can ketonize enol compounds, thereby providing evidence for the presence of an enediolate intermediate. The characterization of FG41 MSAD uncovered an activity it shares with MSAD. FG41 MSAD catalyzes the hydration of 2-oxo-3-pentynoate, but at a rate that is 50-fold less efficient than that of MSAD (as assessed by kcat/Km values). Mutagenesis studies of FG41 MSAD revealed that a single mutation resulted in a 8-fold increase in the activity. The characterization of Cg10062 and attempts to enhance the low-level cis-CaaD activity demonstrated the need for a bacterial screen that could screen a library of mutants. The resulting bacterial screen could be used to screen other members of the superfamily for dehalogenase activity. An in-depth exploration of the Cg10062 and FG41 MSAD activities may lead to a better understanding of the mechanism of cis-CaaD and MSAD and further delineate the evolutionary pathway for the tautomerase superfamily. / text
76

Effects of orally administered spermidine on absorptive enzyme and nutrient transporter gene expression in the rat small intestine during postnatal development

Searles, Lynne E. (Lynne Elizabeth) January 1995 (has links)
The developmental profiles of mRNA and protein expression for ornithine decarboxylase (ODC), the Na$ sp+$-dependent glucose co-transporter (SGLT1), sucrase isomaltase (SI), and the Na$ rm sp+K sp+$ ATPase $ alpha sb1$ and $ beta sb1$ subunit isoforms in the postnatal rat small intestine, as well as the effects of exogenous spermidine on their precocious development, were examined. Postnatal age had a significant effect with all enzymes and the nutrient transporter maturing around weaning. Consecutive exposure to exogenous spermidine during suckling precociously induced ODC mRNA, SI protein, and SGLT1 gene expression in the proximal and distal small intestine. Levels of Na$ rm sp+K sp+$ ATPase $ alpha sb1$ and $ beta sb1$ subunit isoform mRNA were precociously induced in the proximal small intestine only. These findings show that exposure to exogenous spermidine can promote precocious alterations in intestinal enzyme and nutrient transporter expression; however, it appears that spermidine must be continuously supplied for these alterations to persist in suckling rats.
77

Engineering Allium White Rot Disease Resistance in Allium Species and Tobacco Model Species

Glue, Joshua Barnaby January 2009 (has links)
Allium white rot (AWR) is a soilborne disease that seriously damages commercial cultivation of onion (Allium cepa) and garlic (Allium sativum) crops. The disease has been found everywhere onions are cultivated and at present no system of control has been found that fully prevents the occurrence of the disease. The fungus responsible for the disease, Sclerotium cepivorum, uses oxalic acid to kill Allium bulb and root tissue in growing onion and garlic plants. Research suggests recombinant oxalate oxidase and oxalate decarboxylase enzymes may be able to degrade this acid and confer resistance against pathogens that rely on it, such as Sm. cepivorum or Sclerotinia sclerotiorum. To test the efficacy of these enzymes against white rot pathogens, three transgenes for wheat oxalate oxidase, barley oxalate oxidase and Flammulina oxalate decarboxylase were transformed into onions and garlic by Agrobacterium-mediated transformation. Allium species are highly recalcitrant to transformation, so these three transgenes were also transformed into tobacco to provide fast-recovering, easy to test transformants to assess the efficacy of the transgenes. Transformed garlic and tobacco lines were analysed to assess the integration and expression of the transgenes, then challenged with Sm. cepivorum or Sa. sclerotiorum, respectively, to assess the bioactivity of recombinant wheat oxalate oxidase, barley oxalate oxidase, and Flammulina oxalate decarboxylase against oxalic acid-dependent pathogens. Results show that one line of tobacco expressing the Flammulina oxalate decarboxylase enzyme was found to be consistently resistant to Sclerotinia sclerotiorum. Garlic lines transformed with this transgene failed to display stable transgene expression or disease resistance, possibly due to silencing of the transgene in recovered transformant tissue.
78

Cloning And Characterization Of Streptomyces Clavuligerus Meso-diaminopimelate Decarboxylase (lysa) Gene

Yagcioglu, Cigdem 01 September 2004 (has links) (PDF)
In Streptomyces clavuligerus, the route to the biosynthesis of &amp / #945 / -aminoadipic acid (&amp / #945 / -AAA) represents an important primary metabolic pathway providing carbon flux to the synthetases of antibiotic formation. This carbon flow comes through the lysine-specific branch of the aspartate pathway and is rate limiting in the formation of cephamycin C, a second generation cephalosporin produced by this organism. In this study, the lysA gene which encodes for an important key enzyme of aspartate pathway / meso-diaminopimelic acid (DAP) decarboxylase (E.C.4.1.1.20) catalyzing the conversion of diaminopimelate to lysine was cloned and characterized for the first time from S. clavuligerus NRRL 3585. The attempts to clone the gene by constructing libraries of S. clavuligerus genomic DNA and screening of the libraries either by homologous probing or complementation approach gave no positive results. Then, PCR-based cloning was taken as the approach and the gene was amplified with PCR using the primers derived from the conserved sequences of lysA genes in two fragments (620 and 983 bp) which had overlapping regions. Fragments were then cloned and nucleotide sequencing revealed a complete open reading frame (ORF) encoding a protein of 463 aa (Mr 49, 907). The GC content of the gene was identified as 70.98 %. The gene sequence showed 83 % identity to the sequence of S. coelicolor lysA gene and 81 % identity to S. avermitilis lysA gene. By comparing the amino acid sequence of this protein to those available in database, the sites of the enzyme important for catalysis were identified.
79

Engineering Allium White Rot Disease Resistance in Allium Species and Tobacco Model Species

Glue, Joshua Barnaby January 2009 (has links)
Allium white rot (AWR) is a soilborne disease that seriously damages commercial cultivation of onion (Allium cepa) and garlic (Allium sativum) crops. The disease has been found everywhere onions are cultivated and at present no system of control has been found that fully prevents the occurrence of the disease. The fungus responsible for the disease, Sclerotium cepivorum, uses oxalic acid to kill Allium bulb and root tissue in growing onion and garlic plants. Research suggests recombinant oxalate oxidase and oxalate decarboxylase enzymes may be able to degrade this acid and confer resistance against pathogens that rely on it, such as Sm. cepivorum or Sclerotinia sclerotiorum. To test the efficacy of these enzymes against white rot pathogens, three transgenes for wheat oxalate oxidase, barley oxalate oxidase and Flammulina oxalate decarboxylase were transformed into onions and garlic by Agrobacterium-mediated transformation. Allium species are highly recalcitrant to transformation, so these three transgenes were also transformed into tobacco to provide fast-recovering, easy to test transformants to assess the efficacy of the transgenes. Transformed garlic and tobacco lines were analysed to assess the integration and expression of the transgenes, then challenged with Sm. cepivorum or Sa. sclerotiorum, respectively, to assess the bioactivity of recombinant wheat oxalate oxidase, barley oxalate oxidase, and Flammulina oxalate decarboxylase against oxalic acid-dependent pathogens. Results show that one line of tobacco expressing the Flammulina oxalate decarboxylase enzyme was found to be consistently resistant to Sclerotinia sclerotiorum. Garlic lines transformed with this transgene failed to display stable transgene expression or disease resistance, possibly due to silencing of the transgene in recovered transformant tissue.
80

Enzymatic C-C bond formation using thiamine diphosphate dependent enzymes in a solid gas bioreactor /

Mikolajek, Renaud. January 2008 (has links)
Zugl.: Aachen, Techn. Hochsch., Diss., 2008.

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