Browsing by Subject "Mycobacterium tuberculosis"
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Item Structural and Functional Studies of Mycothiol Biosynthesis Precursor Enzyme in Mycobacterium tuberculosis(2012-10-19) Zhu, Wan WenMshA is a glycosyltransferase that synthesizes the precursor of mycothiol, a low-molecular-weight thiol found exclusively in Actinomycetes, including the virulent pathogen Mycobacterium tuberculosis (Mtb). The structure of MshA from Mtb (herein coined as TbMshA) and its complex with uridine diphosphate N-acetyl-glucosamine (UDP-GlcNAc) have been solved to resolutions of 2.32 A and 2.89 A respectively. Both structures form two monomers in the asymmetric unit cell and exhibit typical beta/alpha/beta Rossmann folds. Upon binding of UDP-GlcNAc, the C-terminal domain of TbMshA undergoes conformational changes in order to interact with UDP-GlcNAc at the binding site. In addition, ligand-bound TbMshA structure enables the identification of critical residues for enzymatic interactions, especially the residue Glu-353 (E353) at the active site that is believed to serve as a nucleophile in the sugar transfer of TbMshA. In order to verify this, a mutant of TbMshA with a single amino acid mutation from glutamate to glutamine at residue 353 is generated. The mutant (E353Q) has shown reduced enzyme activity by more than four-fold compared to the wild-type TbMshA (Vmax for wild-type is 0.17 plus/minus 0.02 microM sec^-1, whereas Vmax for E353Q is 0.04 plus/minus 0.01 microM sec-1). The kcat/Km for wild-type TbMshA (3.5 plus/minus 1.1 * 10^3 M^-1 sec^-1) is an order of magnitude higher than that of the mutant (0.3 plus/minus 0.1 * 10^3 M^-1 sec^-1), indicating the catalytic efficiency is greatly suppressed by the mutation. Mass spectrometry data also reveals that E353Q is unable to form the product of the reaction catalyzed by the wild-type TbMshA. These findings suggest the important role of Glu-353 in the structure and activity of TbMshA.Item Structural Studies and Evaluation of Inhibitors of Mycobacterium tuberculosis H37Rv Shikimate Dehydrogenase (MtSDH)(2014-03-21) Lalgondar, MallikarjunShikimate dehydrogenase (SDH) is a reversible enzyme catalyzing the reduction of 3-dehydroshikimate (3DHS) to shikimate (SKM) utilizing NADPH cofactor in the shikimate pathway, a central route for biosynthesis of aromatic amino acids, folates and ubiquinones in microogransims, plants and parasites, which renders the enzymes of this essential pathway as attractive targets for developing antimicrobials, herbicides and antiparasitic agents. In this study, the crystal structure of Mycobacterium tuberculosis SDH (MtSDH) was determined in the apo-form and in complex with a ligand, SKM. The overall structure of MtSDH contains two structural domains with ?/? architecture. The N-terminal substrate binding domain and C-terminal cofactor binding domain are interconnected by two helices forming an active site groove where catalysis occurs. In MtSDH, a series of helices connecting ?10 and ?11 strands replace a long loop found in other known SDH structures and this region may undergo structural changes upon cofactor binding. NADP^(+) was modeled reliably in the cofactor binding site to gain insight into specific interactions. The analysis reveals that NADP(H) binds in anti conformation and in addition to residues in ?basic patch?, Ser125 within the glycine rich loop may interact with the 2'-phosphate of adenine ribose and form a novel cofactor binding microenvironment in SDH family of enzymes. Biochemically, five inhibitors identified previously from a high-throughput enzyme assay screen were evaluated. The IC_(50) values of these compounds range from 2.8-4.6 ?M. Further investigation indicates that these compounds display non-competitive or mixed inhibition mode with both substrate and cofactor. This study is expected to provide better understanding of MtSDH structural features and a framework for rational design of inhibitors based on initially characterized compounds.Item Structural Study of Lipid-binding Proteins(2013-08-09) Tsai, Han-ChunTuberculosis and malaria are among the most deadly infectious diseases in the world. The prevalence in regions without well-established public health causes economical and financial burdens for both society and patients. There is an urgent need to find effective treatments due to the emergence of drug-resistant strains. The aim of the studies reported here was to gain knowledge from the protein structures that can lead to the elimination of these pathogens. In these studies, protein crystallography is the main method used to solve protein structure. Based on the protein structure, we used different methods to characterize the protein function of three lipid-binding proteins (LprG, LprA, and gp232), and to identify potent inhibitors against Plasmodium falciparum enoyl-ACP reductase (PfENR), a drug target protein involved in central lipid metabolism. To characterize the function of two lipid-binding proteins (LprG and LprA), liquid chromatography-mass spectrometry (LC-MS) was used to analyze the ligand extract. In the study of tail fiber protein from mycobacteriophage, we used protein sequence alignment to identify gp232 as a major tail fiber protein, which potentially binds to lipids on the cellular surface of mycobacteria. A pull-down assay and imaging methods (fluorescence microscopy and electron microscopy) were conducted to confirm the function of gp232. In the structural study of PfENR, the structure-activity relationships method was used to find potent inhibitors against PfENR, which would show stronger inhibition than the known inhibitor triclosan. The triclosan-like analogs with modification at the 5-position revealed a new binding site in PfENR that has great potential for improving the potency of inhibition. We found that two inhibitors containing the core structure of piperidine and tetrahydroquinoline reached this new binding site and were 10-fold more potent than triclosan. The structural study of PfENR provides structural insights into the inhibitor-binding site that can lead to the discovery of new drugs. The comprehensive knowledge that we gained from the structural studies of these lipid-binding proteins provide new information that could lead to a greater understanding of pathogen physiology or guide the discovery of effective treatments to eliminate the pathogens.Item The role of CCL5 (RANTES) in the immune response against Mycobacterium tuberculosis in the guinea pig(Texas A&M University, 2005-02-17) Skwor, Troy ArthurTuberculosis is the second leading cause of morbidity and mortality worldwide due to an infectious disease. Development of a new tuberculosis (TB) vaccine would be facilitated by a better understanding of the mechanisms of protection induced by the current TB vaccine, Mycobacterium bovis BCG. Recombinant guinea pig (rgp)CCL5 and anti-rgpCCL5 were developed and characterized. The biological activity of rgpCCL5 was determined in a chemotaxis assay using T lymphocytes and pleural exudate cells. The specificity of rabbit anti-rgpCCL5 polyclonal antibody was confirmed by Western blot. RgpCCL5 was used to stimulate alveolar and peritoneal macrophages in vitro. and cytokine/chemokine gene expression was evaluated using real-time PCR. RgpCCL5 stimulated TNFα, IL-1β, CCL2, and CXCL8 mRNA expression and TNFα protein production (as assessed in the L929 cell bioassay) in macrophages. The effect of BCG-vaccination on CCL5 expression and production in leukocytes infected with M. tuberculosis was examined in vitro and in vivo. Polyclonal anti-rgpCCL5 was used to develop an ELISA assay to quantify gpCCL5 protein levels, and real-time PCR was used to detect CCL5 mRNA. Leukocytes isolated from BCG-vaccinated guinea pigs and infected in vitro with virulent M. tuberculosis demonstrated significantly elevated gpCCL5 mRNA and protein compared to cells from naive animals. The response of gpCCL5 to M. tuberculosis in vivo was studied in tuberculous pleural effusions, where peak levels of CCL5 mRNA and protein were reached at day 4 post-induction. Disease severity, cellular differentiation, histology, and cytokine/chemokine mRNA levels in pleural cells and granulomas were analyzed on day 4 in guinea pigs induced with tuberculous pleurisy and treated with either rgpCCL5 or anti-rgpCCL5 by direct intra-pleural injection. In these studies, neutralizing CCL5 resulted in reduced macrophage accumulation, diminished levels of IFNγ, TNFα, and CCL5 mRNA in pleural effusion cells, and reduced spontaneous lymphocyte proliferation. Together these studies suggest an important role for gpCCL5 in activating leukocytes during M. tuberculosis infection.