title: Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv.
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.
authors.affiliation: 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; Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil.
authors.forename: Priscila Lamb
authors.initials: PL
authors.lastname: Wink
authors.forename: Zilpa Adriana
authors.initials: ZA
authors.lastname: Sanchez Quitian
authors.forename: Leonardo Astolfi
authors.initials: LA
authors.lastname: Rosado
authors.forename: Valnes da Silva
authors.initials: Vda S
authors.lastname: Rodrigues
authors.forename: Guilherme Oliveira
authors.initials: GO
authors.lastname: Petersen
authors.forename: Daniel Macedo
authors.initials: DM
authors.lastname: Lorenzini
authors.forename: Thiago
authors.initials: T
authors.lastname: Lipinski-Paes
authors.forename: Luis Fernando
authors.initials: LF
authors.lastname: Saraiva Macedo Timmers
authors.forename: Osmar Norberto
authors.initials: ON
authors.lastname: de Souza
authors.forename: Luiz Augusto
authors.initials: LA
authors.lastname: Basso
authors.forename: Diogenes Santiago
authors.initials: DS
authors.lastname: Santos
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000032:analysis; D003001:Cloning, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013816:Thermodynamics; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D011994:Recombinant Proteins; D009699:N-Glycosyl Hydrolases; D002118:Calcium
keywords: Mycobacterium tuberculosis; Nucleoside hydrolase; Spectrofluorimetry; Substrate specificity; Thermodynamics; pH-rate profile