PMID:22119138

Citation	Franco, TM, Rostirolla, DC, Ducati, RG, Lorenzini, DM, Basso, LA and Santos, DS (2012) Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain. Arch. Biochem. Biophys. 517:1-11
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.
Links	PubMed Online version:10.1016/j.abb.2011.11.013
Keywords	Adenosine Triphosphatases/metabolism; Amino Acid Sequence; Carbon-Nitrogen Ligases/chemistry; Carbon-Nitrogen Ligases/genetics; Carbon-Nitrogen Ligases/isolation & purification; Carbon-Nitrogen Ligases/metabolism; Cloning, Molecular; Glutaminase/metabolism; Humans; Kinetics; Ligands; Magnesium/metabolism; Models, Molecular; Molecular Sequence Data; Mycobacterium tuberculosis/chemistry; Mycobacterium tuberculosis/enzymology; Mycobacterium tuberculosis/genetics; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sequence Alignment; Titrimetry; Tuberculosis/microbiology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MYCSD:F5Z3Z0		GO:0016740: transferase activity	ECO:0000315:		F		Figure-6	complete CACAO 3776

References

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