Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv.
Arch Biochem Biophys
; 538(2): 80-94, 2013 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-23988349
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Tuberculose
/
Mycobacterium tuberculosis
/
N-Glicosil Hidrolases
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Arch Biochem Biophys
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Brasil