UMD Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/3

New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.

More information is available at Theses and Dissertations at University of Maryland Libraries.

Browse

Subject: search.filters.subject.Enzymology

Search Results

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Investigations of Substrate Recognition of the Biofilm Glycosidase Enzyme Dispersin B
    (2022) Peterson, Alexandra Breslawec; Poulin, Myles B; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Bacterial biofilms, which are comprised of bacterial cells embedded in a thick extracellular polymeric substance (EPS), is a type of survival mechanism used by a variety of medically relevant bacteria to endure harsh conditions, the immune system of the host organism, and medical intervention such as antibiotics. Biofilms confer additional protection to these bacteria, protecting them from a number of stressors, and contribute to the growing problem of antibiotic-resistant infections. Biofilm EPS is comprised of extracellular polysaccharides, proteins, DNA, and other small molecules such as enzymes and nutrients, and the strength and structure of biofilms are often attributed to extracellular polysaccharides such as poly-β-D-(1→6)-N-acetyl-glucosamine (PNAG). Glycoside hydrolase enzymes that are produced as part of the biofilm’s life cycle are being explored as possible anti-biofilm compounds, due to their ability to destabilize biofilms through degradation of the polysaccharide components. The enzyme Dispersin B (DspB), a family 20 glycoside hydrolase produced by Aggregatibacter actinomycetemcomitans, hydrolyzes partially de-N-acetylated PNAG (dPNAG), and shows promise as a potential anti-biofilm agent. Here, we use a variety of techniques to investigate the interactions between DspB and PNAG, leading to a greater understanding of the binding interactions and mechanisms used by DspB to hydrolyze PNAG (Chapter 2). First, the activity of DspB on a monosaccharide probe, 4-methylumbelliferone-GlcNAc (4muGlcNAc) was observed over a pH range to determine the ideal conditions for DspB activity (2.2). Specifically acetylated PNAG trisaccharide analogs were then used to determine the substrate specificity of DspB, which supported the existing hypothesis that DspB uses a substrate-assisted mechanism to hydrolyze PNAG (2.4-2.6). These studies also indicated the possibility of electrostatic interactions between anionic amino acids on the binding surface of DspB and cationic deacetylated residues on PNAG that stabilize the substrate-binding interactions and allow for additional cleavage activities of DspB, namely improved cleavage of partially deacetylated PNAG and the ability to perform endo- or exoglycosidic cleavage activity, dependent on the substrate acetylation patterns present (2.5). Mutagenesis of amino acid residues on the binding surface of DspB was performed to investigate these interactions (Chapters 3-4), resulting in the discovery of an improved DspB mutant. This E248Q mutant of DspB also has an improved ability to clear Staphylococcus epidermidis biofilms, indicating that it may have improved anti-biofilm activity (3.3). Finally, a high-throughput assay for anti-PNAG activity has been developed for use with a degenerate DspB mutant library in order to identify additional DspB mutants with improved anti-biofilm activity.
  • Thumbnail Image
    Item
    USE OF 2-DEOXYFLAVIN MONONUCLEOTIDE TO PROBE SUBSTRATE-FLAVIN INTERACTIONS WITHIN IODOTYROSINE DEIODINASE
    (2015) Boucher, Petrina Abiola; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The catalytic versatility of flavins encompasses the ability to catalyze one and two electron redox processes as well as dioxygen activation. In flavoenzymes, protein-cofactor interactions modulate flavin chemistry enabling these enzymes to perform a diverse set of biological roles. Flavin analogs, such as deoxyflavins, provide a convenient method for identifying which polar contacts to FMN are necessary for enzymatic activity. Iodotyrosine deiodinase (IYD) is responsible for the deiodination of byproducts from thyroxine biosynthesis (mono- and diiodotyrosine (MIT and DIT)) in the thyroid. A unique flavoenzyme, IYD, is one of few known aerobic enzymes able to catalysis reductive dehalogenation. Literature precedence for the deiodination mechanism of IYD has not been found. Detection of the flavin semiquinone (Flsq) intermediate during anaerobic reduction of IYD in the presence of substrate suggests deiodination occurs via two single electron transfer processes. Proposed one electron mechanisms for IYD involving formation of a substrate keto-tautomer are supported by the enzyme’s preference for the phenolate form of substrate during binding. Previous analysis of a co-crystal of IYD and MIT showed the involvement of substrate in extensive interactions with the FMN cofactor, including a hydrogen bond between the FMN ribityl 2ʹ-OH and the phenolic OH of MIT. This hydrogen bond has been hypothesized to activate substrate for deiodination. To probe the significance of the polar interaction between the ribityl 2ʹ-OH and the phenolic OH of MIT in IYD, 2-deoxyriboflavin was synthesized and enzymatically phosphorylated using riboflavin kinase. Reconstitution of human IYD (hIYD) with 2-deoxyFMN produced an enzyme with a significantly decreased affinity for MIT. Deiodinase activity was retained in 2-deoxyhIYD with a 10-fold decrease in catalytic efficiency. Detection of the Flsq was not observed during anaerobic reduction of 2-deoxyhIYD in the presence of mono-fluorotyrosine (MFT). The enzyme’s preference for the phenol versus phenolate form of substrate could not be determined due to the low solubility of DIT at concentrations necessary for pH dependent binding analysis. Removal of the ribityl 2ʹ-OH did not support turnover of O-methyl MIT, a substrate analog incapable of undergoing tautomerization.
  • Thumbnail Image
    Item
    Kinetic and Structual Characterization of Glutamine-Dependent NAD Synthetases
    (2010) Resto, Melissa; Gerratana, Barbara; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Multifunctional enzymes catalyzing successive reactions have evolved several mechanisms for the transport of intermediates between active sites. One mechanism, substrate channeling, allows the transport of the intermediate without releasing it into the solvent. Members of the glutamine amidotransferase (GAT) family often utilize substrate channeling for the transport of intermediates. GAT enzymes hydrolyze glutamine to ammonia, which is transported to an acceptor domain preventing wasteful hydrolysis of glutamine and increasing the efficiency of the reaction. Many GAT enzymes utilize molecular tunnels to shuttle ammonia between active sites. Often GAT enzymes synchronize the active site through conformational changes that occur during catalysis. Glutamine-dependent NAD synthetases are GAT enzymes and catalyze the last step in the biosynthesis of NAD, utilizing nicotinic acid adenine dinucleotide (NaAD), ATP and glutamine. Steady-state kinetic characterizations and stoichiometric analysis of NAD synthetase from Mycobacterium tuberculosis (NAD synthetaseTB) revealed a substrate channeling mechanism for ammonia transport and tight coordination of the active sites resulting in an enzyme that is highly efficient in the use of glutamine. The crystal structure of NAD synthetaseTB has revealed a 40 Å tunnel that connects the active sites and is postulated to play a role in the synchronized activities. Several regions of the enzyme were identified that may be important for regulation, such as the YRE loop which contacts the glutamine active site and key regions of the tunnel. Mutations of tunnel residues, such as D656A, show that interruption of important interactions can result in compromise in transfer of ammonia or active site communication. Phylogenetic analysis revealed that glutamine-dependent NAD synthetases have different levels of regulation. Three groups of enzymes were identified represented by NAD synthetase from M. tuberculosis, S. cerevisiae (NAD synthetaseYeast) and Thermotoga maritima (NAD synthetaseTM). Steady-state kinetic characterizations and stoichiometric analysis of NAD synthetaseTM has revealed a compromised coordination of the active sites compared to the highly synchronized NAD synthetaseTB and the moderate synchronization of NAD synthetaseYeast. Sequence alignment of these groups has allowed identification of residues that line the tunnel that may be responsible for the differences observed in active site coordination and are, therefore, important for active site communication.