Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis.
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 approximately 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.
Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India.
Sangeeta
S
Tiwari
K V Radha
KV
Kishan
Tapan
T
Chakrabarti
Pradip K
PK
Chakraborti
D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D000596:Amino Acids; D017931:DNA Primers; D011994:Recombinant Proteins; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase