Chemicals and Drugs Commons^™

Characterizing And Overcoming Resistance To Aminomethylspectinomycins In Gram-Negative Bacteria, Nisha Das

Theses and Dissertations (ETD)

Spectinomycin (SPC) is a broad-spectrum aminocyclitol antibiotic. Its use in agriculture has led to widespread resistance in enteric bacteria, necessitating the development of more effective analogs. Aminomethyl spectinomycins (amSPC) are modified spectinomycins with increased potency against many bacterial species. These species include Legionella pneumophila, which harbors a chromosomally encoded aminoglycoside modifying enzyme (AME). In this study, we follow up on this observation and examine the extent to which the amSPCs are substrates for AMEs through adenylation (ANTs) and phosphorylation (APH). APH(9)-Ia and ANT(3")(9) were expressed in E. coli BL21(DE3) and purified using the Ni-affinity chromatography. The ability of AMEs to …

Interaction Between Two E3 Ligases, Nedd8ylated Cullin And Hhari, Kheewoong Baek

Theses and Dissertations (ETD)

RBR (RING1-in between RING-RING2) is a special type of E3 ubiquitin ligase containing three zinc-binding RING (Really Interesting New Gene) domains, while adopting mechanisms of HECT (Homologous to E6-AP Carboxyl Terminus) for substrate ubiquitination. Most well known RBRs include Parkin and HOIP, which are associated with Parkinson’s disease and innate immune deficiency. However, it is not well known how the RBR proteins gain activity, as they are known to be autoinhibited. Here I show that a specific F430A, E431A, E503A triple mutation of RBR protein HHARI (Human homologue of Ariadne) and its interaction with NEDD8ylated cullin RING ligase can both …

The Molecular Basis Of Fitness And Transmissibility Of Neuraminidase Inhibitor Resistant Influenza A Viruses, Susu Duan

Theses and Dissertations (ETD)

Neuraminidase (NA) inhibitors including oral oseltamivir and inhaled zanamivir are among the first line of defense against influenza virus infection. Development of resistance to NA inhibitors is a huge drawback for limited options for the control of influenza. During the first decade of NA inhibitor use, the detection rates of resistance to both NA inhibitors had remained low in circulating influenza viruses. However, the 2008~2009 season was marked by a radical increase of prevalence of oseltamvir resistance from <1% to >90% in worldwide surveillance in less than a year. The resistance was solely linked to NA H275Y variants of seasonal H1N1 viruses, …

Insights Into Btb-Cul3 Ubiquitin Ligases From The Structures Of Spop-Substrate Complexes, Min Zhuang

Theses and Dissertations (ETD)

Cullin-Ring ubiquitin ligases (CRLs) are E3 complexes that specifically recognize substrates through substrate adaptors. In the largest CRL subfamily, Cul3 binds a BTB domain, and a protein-interaction domain such as MATH recruits substrates for ubiquitination. Here we present biochemical and structural analyses of the MATH and BTB domain containing protein, SPOP, which regulates diverse signaling pathways. First, we identified a conserved SPOP Binding Consensus (SBC) motif in the transcriptional regulator Ci, the protein phosphatase Puc, and the chromatin component MacroH2A. The SBC motif specifically binds the MATH domain of SPOP, and is required for Puc ubiquitination in vitro and in …

Cdc45 Function Alters Cell Sensitivity To Dna Topoisomerase I Poisons, Cynthia Sue Lancaster

Theses and Dissertations (ETD)

Eukaryotic DNA topoisomerase I (Top1) is a highly conserved enzyme that functions to manage the torsional strain of DNA during cellular processes such as transcription, replication, chromatid condensation and recombination. The enzyme binds duplex DNA and through a series of strand cleavage and religation reactions removes positive or negative supercoils relieving torsional strain. Top1 is the sole cellular target of the anticancer agent camptothecin, which stabilizes the covalent complex. CPT cytotoxicity is S-phase dependent. It has been suggested that the mechanism of this S-phase toxicity is due to the advancing replication forks either colliding with the stabilized drug-enzyme-DNA intermediate or …

Enzyme Architecture And Flexibility Affect Dna Topoisomerase I Function, Mariè Van Der Merwe

Theses and Dissertations (ETD)

DNA topoisomerase I (Top1) is a highly conserved enzyme composed of four domains: a positively charged N-terminus; a DNA binding/core domain that circumscribes duplex DNA; a non-conserved linker domain; and a C-terminal/catalytic domain. Top1 catalyzes changes in DNA topology by transient cleavage of a single DNA strand and the concomitant formation of a phosphotyrosyl linkage between the enzyme and the 3’ DNA end. This covalent Top1-DNA complex is the binding site for camptothecin (CPT), which selectively inhibits religation of the cleaved DNA strand. CPT binding stabilizes the covalent complex, while the collision of replication forks with CPT-Top1-DNA adducts produces DNA …

Characteristics Of The Recovery Of The Coenzyme A-Synthesizing Protein Complex From Saccharomyces Cerevisiae, Stanley Joseph Tarnowski Jr.

Theses and Dissertations (ETD)

A multienzyme complex contained in Bakers' yeast (Saccharomyces cerevisiae) which synthesizes CoA has been named the coenzyme A-synthesizing protein complex (CoA-SPC). The CoA-SPC has been shown to be insoluble in the crude Bakers' yeast cell lysate formed by exposing the yeast cell to ether and dry ice. Only after solubilization has this multienzyme complex been shown to catalyze the formation of bound dephospho-CoA utilizing the substrates adenosine triphosphate, D-pantothenic acid and L-cysteine. A low molecular weight component or components of the soluble fraction of the yeast cell and chloride ion appears to be responsible for the solubilization of CoA-SPC. This …

The Purification, Properties And Subunit Structure Of Glycerol Dehydrogenase, Michael James Barrett

Theses and Dissertations (ETD)

The inducible, NAD-linked glycerol dehydrogenase (E.C. 1.1.1.6) of a guanine requiring mutant of A. aerogenes has been purified to homogeneity. The molecular weight of the pure enzyme was found to be 3.4 x 10⁵ daltons. The sedimentation coefficient of the enzyme was 10.7 x 10^-13 sec.^-1. The diffusion constant was 3.07 x 10^-7 cm²/sec.

The partial specific volume calculated from the amino acid composition was 0.72 ml/g. The following kinetic parameters were determined; K_m glycerol, 2.4 x 10^-3 m, K_m NAD, 2.8 x 10^-4 M, K_m dihydroxyacetone, 5.1 …