Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis

Sangeeta Tiwari, K. V. Radha Kishan, Tapan Chakrabarti, Pradip K. Chakraborti

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Nucleoside diphosphate kinase (NdK) is a ubiquitous enzyme in both prokaryotes and eukaryotes and is primarily involved in the maintenance of cellular nucleotide pools. We have cloned ndk from Mycobacterium tuberculosis strain H37Ra and expressed it in Escherichia coli as a fusion protein with glutathione S-transferase. The purified protein, following thrombin cleavage and gel permeation chromatography, was found to be hexameric with a monomeric unit molecular mass of ∼16.5 kDa. The protein exhibited nucleotide binding, divalent cation-dependent autophosphorylation, and phosphate transfer ability from nucleoside triphosphate to nucleoside diphosphate. Although UDP inhibited the catalytic activity of the recombinant protein, the classic inhibitors, like cromoglycate, 5′-adenosine 3′-phosphate, and adenosine 3′-phosphate 5′-phosphosulfate, had no effect on the activity. Among three histidine residues in the protein, His-117 was found to be essential for autophosphorylation. However, in subsequent phosphate transfer, we observed that His-53 had a significant contribution. Consistent with this observation, substitution of His-53 with either Ala or Gln affected the ability of the recombinant protein to complement NdK function in Pseudomonas aeruginosa. Furthermore, mutational analysis established critical roles for Tyr-50 and Arg-86 of the M. tuberculosis protein in maintaining phosphotransfer ability.

Original languageEnglish (US)
Pages (from-to)43595-43603
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number42
DOIs
StatePublished - Oct 15 2004
Externally publishedYes

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Nucleoside-Diphosphate Kinase
Mycobacterium tuberculosis
Amino Acids
Proteins
Nucleosides
Recombinant Proteins
Nucleotides
Phosphoadenosine Phosphosulfate
Phosphates
Cromolyn Sodium
Uridine Diphosphate
Diphosphates
Divalent Cations
Gel permeation chromatography
Molecular mass
Adenosine Monophosphate
Glutathione Transferase
Eukaryota
Histidine
Thrombin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis. / Tiwari, Sangeeta; Radha Kishan, K. V.; Chakrabarti, Tapan; Chakraborti, Pradip K.

In: Journal of Biological Chemistry, Vol. 279, No. 42, 15.10.2004, p. 43595-43603.

Research output: Contribution to journalArticle

Tiwari, Sangeeta ; Radha Kishan, K. V. ; Chakrabarti, Tapan ; Chakraborti, Pradip K. / Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 42. pp. 43595-43603.
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