Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv |
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Authors: | Priscila Lamb Wink Zilpa Adriana Sanchez Quitian Leonardo Astolfi Rosado Valnes da Silva Rodrigues Júnior Guilherme Oliveira Petersen Daniel Macedo Lorenzini Thiago Lipinski-Paes Luis Fernando Saraiva Macedo Timmers Osmar Norberto de Souza Luiz Augusto Basso Diogenes Santiago Santos |
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Institution: | 1. Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), 6681/92-A Av. Ipiranga, 90619-900 Porto Alegre, RS, Brazil;2. Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil;3. Laboratório de Bioinformática, Modelagem e Simulação de Biossistemas (LABIO), Faculdade de Informática, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil |
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Abstract: | 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. |
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Keywords: | Mycobacterium tuberculosis Nucleoside hydrolase Substrate specificity Thermodynamics pH-rate profile Spectrofluorimetry |
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