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J. Biol. Chem., Vol. 282, Issue 16, 11866-11873, April 20, 2007

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The Helicobacter pylori Amidotransferase GatCAB Is Equally Efficient in Glutamine-dependent Transamidation of Asp-tRNA^Asn and Glu-tRNA^Gln*

Kelly Sheppard¹, Pierre-Marie Akochy¹², Juan C. Salazar³, and Dieter Söll⁴

From the Departments of Molecular Biophysics and Biochemistry and Chemistry, Yale University, New Haven, Connecticut 06520-8114

The amide aminoacyl-tRNAs, Gln-tRNA^Gln and Asn-tRNA^Asn, are formed in many bacteria by a pretranslational tRNA-dependent amidation of the mischarged tRNA species, Glu-tRNA^Gln or Asp-tRNA^Asn. This conversion is catalyzed by a heterotrimeric amidotransferase GatCAB in the presence of ATP and an amide donor (Gln or Asn). Helicobacter pylori has a single GatCAB enzyme required in vivo for both Gln-tRNA^Gln and Asn-tRNA^Asn synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNA^Asn and Glu-tRNA^Gln with similar efficiency (k_cat/K_m of 1368.4 s^-1/mM and 3059.3 s^-1/mM respectively). The essential glutaminase activity of the enzyme is a property of the A-subunit, which displays the characteristic amidase signature sequence. Mutations of the GatA catalytic triad residues (Lys⁵², Ser¹²⁸, Ser¹⁵²) abolished glutaminase activity and consequently the amidotransferase activity with glutamine as the amide donor. However, the latter activity was rescued when the mutant enzymes were presented with ammonium chloride. The presence of Asp-tRNA^Asn and ATP enhances the glutaminase activity about 22-fold. H. pylori GatCAB uses the amide donor glutamine 129-fold more efficiently than asparagine, suggesting that GatCAB is a glutamine-dependent amidotransferase much like the unrelated asparagine synthetase B. Genomic analysis suggests that most bacteria synthesize asparagine in a glutamine-dependent manner, either by a tRNA-dependent or in a tRNA-independent route. However, all known bacteria that contain asparagine synthetase A form Asn-tRNA^Asn by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism.

Received for publication, January 16, 2007 , and in revised form, February 20, 2007.

^* This work was supported in part by National Institute of General Medical Sciences Grant GM22854 and Dept. of Energy Grant DE-FG02-98ER20311. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ Both authors contributed equally to this report.

² Present address: Centre de Recherche en Infectiologie, CHU Laval, Sainte-Foy, Québec, Canada.

³ Present address: Instituto de Ciencias Biomedicas, Universidad de Chile, Santiago, Chile.

⁴ To whom correspondence should be addressed: Dept. of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Ave, New Haven, CT 06520-8114. Tel.: 203-432-6204; Fax: 203-432-6202; E-mail: dieter.soll{at}yale.edu.

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