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J. Biol. Chem., Vol. 279, Issue 42, 43595-43603, October 15, 2004

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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, and Pradip K. Chakraborti

From the 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 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.

Received for publication, February 16, 2004 , and in revised form, July 28, 2004.

^* This work was supported in part by a research grant from the Department of Biotechnology, New Delhi, India, as part of a National Bioscience Award for Career Development to one of the authors (to P. K. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Senior Research Fellowship from the Council of Scientific and Industrial Research, New Delhi, India.

To whom correspondence should be addressed. Tel.: 91-172-269-5215 (ext. 452); Fax: 91-172-269-0585; E-mail: pradip{at}imtech.res.in.

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