TY - JOUR
T1 - Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis
AU - Tiwari, Sangeeta
AU - Radha Kishan, K. V.
AU - Chakrabarti, Tapan
AU - Chakraborti, Pradip K.
PY - 2004/10/15
Y1 - 2004/10/15
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M401704200
DO - 10.1074/jbc.M401704200
M3 - Article
C2 - 15302878
AN - SCOPUS:6344237576
SN - 0021-9258
VL - 279
SP - 43595
EP - 43603
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 42
ER -