Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv
| Autor: | Diógenes Santiago Santos, Guilherme Oliveira Petersen, Osmar Norberto de Souza, Thiago Lipinski-Paes, Zilpa Adriana Sanchez Quitian, Luis Fernando Saraiva Macedo Timmers, Luiz Augusto Basso, Leonardo Astolfi Rosado, Daniel Macedo Lorenzini, Valnes da Silva Rodrigues, Priscila Lamb Wink |
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| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular pH-rate profile Protein Conformation Stereochemistry Molecular Sequence Data Biophysics Guanosine Nucleoside hydrolase Biochemistry Substrate Specificity chemistry.chemical_compound Hydrolase medicine Humans Tuberculosis Amino Acid Sequence Cloning Molecular Inosine N-Glycosyl Hydrolases Molecular Biology Hypoxanthine Isothermal titration calorimetry Uracil Mycobacterium tuberculosis Hydrogen-Ion Concentration Recombinant Proteins Uridine Kinetics chemistry Thermodynamics Calcium Spectrofluorimetry Sequence Alignment Nucleoside medicine.drug |
| Zdroj: | Archives of Biochemistry and Biophysics. 538:80-94 |
| ISSN: | 0003-9861 |
| DOI: | 10.1016/j.abb.2013.08.011 |
| Popis: | Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease’s causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli’s nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH. |
| Databáze: | OpenAIRE |
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