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J. Biol. Chem., Vol. 279, Issue 21, 22477-22482, May 21, 2004

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Adenylylation and Catalytic Properties of Mycobacterium tuberculosis Glutamine Synthetase Expressed in Escherichia coli versus Mycobacteria^*

Ranjana Mehta, Josh T. Pearson, Sumit Mahajan, Abhinav Nath, Mark J. Hickey¶, David R. Sherman¶, and William M. Atkins||

From the Departments of ^¶Pathobiology and Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610

Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456–464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially (25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated (30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.

Received for publication, February 13, 2004 , and in revised form, March 1, 2004.

^* This work was supported by National Institutes of Health Grants AI49780 and GM32165 (mass spectrometry) and National Institutes of Health Training Grant T32 GM07750 (to J. T. P.). 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.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed. Tel.: 206-68-0379; Fax: 206-685-3252; E-mail: winky{at}u.washington.edu.

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