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Biotechnology and Applied Biochemistry (2003) 38, (169–174) (Printed in Great Britain)

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Nucleoside diphosphate kinase-like activity in adenylate kinase of Mycobacterium tuberculosis

Laxman S. Meena*†, Puneet Chopra*, R. S. Bedwal† and Yogendra Singh*¹

*Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007, India, and †Department of Zoology, University of Rajasthan, Jaipur, India

Key words: fusion protein, nucleoside monophosphate kinase, nucleotide metabolism, site-directed mutagenesis, tuberculosis.

Abbreviations used: Ak, adenylate kinase; GST, glutathione S-transferase; LB, Luria–Bertani; Ndk, nucleoside diphosphate kinase; PEI, polyethyleneimine.

¹To whom correspondence should be addressed (e-mail ysingh@igib.res.in).

Ak (adenylate kinase) is a ubiquitous enzyme that catalyses a reversible high-energy phosphoryl-transfer reaction between ATP and AMP to form ADP. In the present study, the Ak gene (adk) of Mycobacterium tuberculosis was cloned, expressed in Escherichia coli and purified as a glutathione S-transferase fusion protein. Purified Ak converted AMP into ADP in the presence of [g-³²P]ATP or [g-³²P]GTP. Replacement of arginine-88 of adk with glycine resulted in the loss of enzymic activity. The purified protein also showed Ndk (nucleoside diphosphate kinase)-like activity as it transferred terminal phosphate from [g-³²P]ATP to all nucleoside diphosphates, converting them into corresponding triphosphates. However, Ndk-like activity of Ak was not observed with [g-³²P]GTP. Immunoblot analysis of various cellular fractions of M. tuberculosis H₃₇Rv revealed that Ak is a cytoplasmic protein. The dual activity of Ak as both nucleoside mono- and di-phosphate kinases suggested that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism.

Received 17 December 2002/14 April 2003; accepted 9 June 2003

Published as Immediate Publication 9 June 2003, DOI 10.1042/BA20020122

© 2003 Portland Press Ltd