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

Tathyana Mar A. Franco, Diana C. Rostirolla, Rodrigo G. Ducati, Daniel M. Lorenzini, Luiz A. Basso, Diógenes S. Santos

Research output: Contribution to journalArticle

8 Scopus citations

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, 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.

Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume517
Issue number1
DOIs
Publication statusPublished - Jan 1 2012

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Keywords

  • Cooperative kinetics
  • Guanosine monophosphate synthetase
  • Protein function
  • Recombinant protein
  • Steady-state kinetics
  • Tuberculosis

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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