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J. Biol. Chem., Vol. 283, Issue 42, 28464-28470, October 17, 2008

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Importance of the Conserved Residues in the Peptidoglycan Glycosyltransferase Module of the Class A Penicillin-binding Protein 1b of Escherichia coli^*

Mohammed Terrak, A Research Associate of the National Fond for Scientific Research (FNRS, Belgium)¹, Eric Sauvage, Adeline Derouaux, Dominique Dehareng, Ahmed Bouhss, Eefjan Breukink^¶, Sylvie Jeanjean, and Martine Nguyen-Distèche

From the Centre d'Ingénierie des Protéines, Institut de Chimie, Université de Liège, B6a, B-4000 Sart-Tilman, Belgium, the Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université Paris-Sud 11, 91405 Orsay, France, and the ^¶Department of Biochemistry of Membranes, Bijvoet Center for Biomolecular Research, Institute of Biomembranes, Utrecht University, Padualaan, 8, 3584 CH, Utrecht, The Netherlands

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu²³³ of motif 1 and Glu²⁹⁰ of motif 3, the residues Phe²³⁷ and His²⁴⁰ of motif 1 and Gly²⁶⁴, Thr²⁶⁷, Gln²⁷¹, and Lys²⁷⁴ of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp²³⁴, Phe²³⁷, His²⁴⁰, Thr²⁶⁷, and Gln²⁷¹ are proposed to maintain the structure of the active site and the positioning of the catalytic Glu²³³.

Received for publication, April 28, 2008 , and in revised form, June 23, 2008.

^* This work was supported by grants from the European Commission within the "EUR-INTAFAR" (Inhibition of New Targets for Fighting Antibiotic Resistance) (LSHM-CT-2004-512138) network, the Belgian programme of Interuniversity Poles of Attraction (IAP) initiated by the Federal Office for Scientific Technical and Cultural Affairs (IAP Number 6/19), the Actions de Recherche Concertées (Grant 03/08-297), and the Fonds National de la Recherche Scientifique (Fonds de la Recherche Fondamentale Collective d'initiative des chercheurs (FRFC) Numbers 2.4506.08 and 2.4511.06). 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 a supplemental table.

¹ Tel.: 32-4-3663397; Fax: 32-4-3663364; E-mail: mterrak{at}ulg.ac.be.

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