title: Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain.
abstract: Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PP(i), and glutamate. XMP, NH(4)(+), and Mg(2+) displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH(3) and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PP(i) is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family.
authors: Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil.
authors: Tathyana Mar A
authors: TM
authors: Franco
authors: Diana C
authors: DC
authors: Rostirolla
authors: Rodrigo G
authors: RG
authors: Ducati
authors: Daniel M
authors: DM
authors: Lorenzini
authors: Luiz A
authors: LA
authors: Basso
authors: Diógenes S
authors: DS
authors: Santos
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D005972:Glutaminase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017186:Titrimetry; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D005972:Glutaminase; D000251:Adenosine Triphosphatases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing); D008274:Magnesium