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J. Biol. Chem., Vol. 282, Issue 42, 30414-30422, October 19, 2007

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Unexpected Inhibition of Peptidoglycan LD-Transpeptidase from Enterococcus faecium by the -Lactam Imipenem^*

From the ^aINSERM, U872, LRMA Pôle 4, Equipe 12 F-75006 Paris, France, ^bCentre de Recherche des Cordeliers, Université Pierre et Marie Curie, UMR S872, F-75006, Paris, France, ^cUniversité Paris Descartes, UMR S872, F-75006 Paris, France, ^dAssistance Publique-Hospitaux de Paris, Hôpital Européen Georges Pompidou, F-75015 Paris, France, ^eCNRS, UMR 5153, F-75231 Paris, France, ^fINSERM, U565, F-75231 Paris, France, ^gMuseum National d'Histoire Naturelle, Mass Spectrometry Facility, USM 0503, F-75231 Paris, France, ^hMuséum National d'Histoire Naturelle, USM0502, Plateforme de Spectrométrie de Masse et de Protéomique du Muséum, Département Recherche Développement et Diversité Moléculaire, F-75005 Paris, France, ⁱCNRS, UMR 8041, F-75005 Paris, France, and ^jMedical and Research Services, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106

The -lactam antibiotics mimic the D-alanyl⁴-D-alanine⁵ extremity of peptidoglycan precursors and act as "suicide" substrates of the DD-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the DD-transpeptidases by the LD-transpeptidase of Enterococcus faecium (Ldt_fm) leads to high level resistance to ampicillin. Ldt_fm is specific for the L-lysyl³-D-alanine⁴ bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine⁵. This specificity was proposed to account for resistance, because the substrate of Ldt_fm does not mimic -lactams in contrast to the D-alanyl⁴-D-alanine⁵ extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a -lactam of the carbapenem class, totally inhibited Ldt_fm at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldt_fm is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldt_fm by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldt_fm containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldt_fm is likely to involve rupture of the -lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of -lactams is not restricted to transpeptidases of the DD-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities.

Received for publication, May 24, 2007 , and in revised form, July 10, 2007.

^* This work was supported by the Fondation pour la Recherche Médicale (Equipe FRM 2006 (Grant DEQ200661107918)) and by NIAID, National Institutes of Health Grant R01 AI45626. 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.

¹ To whom correspondence should be addressed. Tel.: 33-1-43-25-00; Fax: 33-1-43-25-68-12; E-mail: michel.arthur{at}bhdc.jussieu.fr.

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