Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain

• Published in: Archives of biochemistry and biophysics Vol. 517; no. 1; pp. 1 - 11
• Main Authors: Franco, Tathyana Mar A., Rostirolla, Diana C., Ducati, Rodrigo G., Lorenzini, Daniel M., Basso, Luiz A., Santos, Diógenes S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2012
• Subjects: adenosine diphosphate; Adenosine Triphosphatases - metabolism; adenosine triphosphate; adults; Amino Acid Sequence; ammonia; ammonium compounds; calorimetry; Carbon-Nitrogen Ligases - chemistry; Carbon-Nitrogen Ligases - genetics; Carbon-Nitrogen Ligases - isolation & purification; Carbon-Nitrogen Ligases - metabolism; Cloning, Molecular; Cooperative kinetics; glutamic acid; Glutaminase - metabolism; glutamine; GMP synthase (glutamine-hydrolysing); guanosine monophosphate; Guanosine monophosphate synthetase; Humans; hydrolysis; Kinetics; Ligands; magnesium; Magnesium - metabolism; mass spectrometry; Models, Molecular; Molecular Sequence Data; Mycobacterium tuberculosis; Mycobacterium tuberculosis - chemistry; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; neonates; operon; Protein Binding; Protein function; Protein Multimerization; Protein Structure, Tertiary; Recombinant protein; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Alignment; Steady-state kinetics; titration; Titrimetry; Tuberculosis; Tuberculosis - microbiology; vaccines; Cooperative kinetics; Tuberculosis; Recombinant protein; Steady-state kinetics; Protein function; Guanosine monophosphate synthetase
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2011.11.013

[Display omitted] ► The Mycobacterium tuberculosis guaA gene encodes a homodimeric GMP synthetase (MtGMPS). ► The two-domain type MtGMPS exhibits positive homotropic cooperativity for XMP and NH4+. ► Mg2+ saturation curve is sigmoidal and there may be an additional binding site for this metal. ► Activity of ATPPase domain is independent of GATase domain. ► Substrate binding is random and PPi is the last product released. 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, PPi, and glutamate. XMP, NH4+, and Mg2+ 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 NH3 and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PPi 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.