Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis. 2004

Sangeeta Tiwari, and K V Radha Kishan, and Tapan Chakrabarti, and Pradip K Chakraborti
Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India.

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D009169 Mycobacterium tuberculosis A species of gram-positive, aerobic bacteria that produces TUBERCULOSIS in humans, other primates, CATTLE; DOGS; and some other animals which have contact with humans. Growth tends to be in serpentine, cordlike masses in which the bacilli show a parallel orientation. Mycobacterium tuberculosis H37Rv
D009701 Nucleoside-Diphosphate Kinase An enzyme that is found in mitochondria and in the soluble cytoplasm of cells. It catalyzes reversible reactions of a nucleoside triphosphate, e.g., ATP, with a nucleoside diphosphate, e.g., UDP, to form ADP and UTP. Many nucleoside diphosphates can act as acceptor, while many ribo- and deoxyribonucleoside triphosphates can act as donor. EC 2.7.4.6. Deoxynucleoside Diphosphate Kinases,GDP Kinase,Nucleoside Diphosphokinases,Nucleoside-Diphosphate Kinases,Diphosphate Kinases, Deoxynucleoside,Diphosphokinases, Nucleoside,Kinase, GDP,Kinase, Nucleoside-Diphosphate,Kinases, Deoxynucleoside Diphosphate,Kinases, Nucleoside-Diphosphate,Nucleoside Diphosphate Kinase,Nucleoside Diphosphate Kinases
D010766 Phosphorylation The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety. Phosphorylations
D010770 Phosphotransferases A rather large group of enzymes comprising not only those transferring phosphate but also diphosphate, nucleotidyl residues, and others. These have also been subdivided according to the acceptor group. (From Enzyme Nomenclature, 1992) EC 2.7. Kinases,Phosphotransferase,Phosphotransferases, ATP,Transphosphorylase,Transphosphorylases,Kinase,ATP Phosphotransferases
D011487 Protein Conformation The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain). Conformation, Protein,Conformations, Protein,Protein Conformations
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli

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