TY - JOUR
T1 - Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain
AU - Franco, Tathyana Mar A.
AU - Rostirolla, Diana C.
AU - Ducati, Rodrigo G.
AU - Lorenzini, Daniel M.
AU - Basso, Luiz A.
AU - Santos, Diógenes S.
N1 - Funding Information:
This work was supported by funds of Decit/SCTIE/MS-MCT-CNPq-FNDCT-CAPES to National Institute of Science and Technology on Tuberculosis (INCT-TB) to D.S.S. and L.A.B. L.A.B. and D.S.S. also acknowledge financial support awarded by FAPERGS-CNPq-PRONEX-2009 . D.S.S. ( CNPq , 304051/1975-06 ) and L.A.B. (CNPq, 520182/99-5 ) and are Research Career Awardees of the National Research Council of Brazil (CNPq). T.M.A.F., D.C.R., R.G.D., and D.M.L. acknowledge scholarships awarded by CNPq.
PY - 2012/1/1
Y1 - 2012/1/1
N2 - 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, NH4+, 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.
AB - 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, NH4+, 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.
KW - Cooperative kinetics
KW - Guanosine monophosphate synthetase
KW - Protein function
KW - Recombinant protein
KW - Steady-state kinetics
KW - Tuberculosis
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U2 - 10.1016/j.abb.2011.11.013
DO - 10.1016/j.abb.2011.11.013
M3 - Article
C2 - 22119138
AN - SCOPUS:84155164743
SN - 0003-9861
VL - 517
SP - 1
EP - 11
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -