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. (2004) Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from mycobacterium tuberculosis Journal of Biological Chemistry, 279 (42). pp. 43595-43603. ISSN 0021-9258

Full text not available from this repository.

Official URL: http://www.jbc.org/content/279/42/43595.abstract

Related URL: http://dx.doi.org/10.1074/jbc.M401704200

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.

Item Type:Article
Source:Copyright of this article belongs to The American Society for Biochemistry and Molecular Biology.
ID Code:60363
Deposited On:08 Sep 2011 14:37
Last Modified:08 Sep 2011 14:37

Repository Staff Only: item control page