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J. Biol. Chem., Vol. 283, Issue 10, 6402-6417, March 7, 2008

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Characterization of tRNA-dependent Peptide Bond Formation by MurM in the Synthesis of Streptococcus pneumoniae Peptidoglycan^*

Adrian J. Lloyd¹, Andrea M. Gilbey, Anne M. Blewett, Gianfranco De Pascale, Ahmed El Zoeiby^¶, Roger C. Levesque^¶, Anita C. Catherwood, Alexander Tomasz^||, Timothy D. H. Bugg, David I. Roper, and Christopher G. Dowson

From the Departments of Biological Sciences and Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom, the ^¶Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Faculté de Médecine, Pavillon Charles-Eugène Marchand, Université Laval, Ste-Foy, Quebec G1K 7P4, Canada, and the ^||Laboratory of Microbiology, The Rockefeller University, New York, New York 10021

MurM is an aminoacyl ligase that adds L-serine or L-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM₁₅₉ alanylated the peptidoglycan -amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM_Pn16 activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM_Pn16 acylation activities were far lower than the alanylation activity of MurM₁₅₉. The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM₁₅₉ compared with MurM_Pn16 bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA^Ala:tRNA^Ser ratio in 159 and Pn16. Kinetic analysis revealed that MurM₁₅₉ acted during the lipid-linked stages of peptidoglycan synthesis, that the D-alanyl-D-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that -carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TC loop stem of the tRNA^Ala. It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of β-lactams for treatment of pneumococcal infection.

Received for publication, September 28, 2007 , and in revised form, December 7, 2007.

^* This work was supported by Wellcome Trust Grant 066443. 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 Materials and Methods, Results, additional references, Figs. S1-S5, and Table S1.

¹ To whom correspondence should be addressed. Tel.: 44-2476-522568; Fax: 44-2476-523701; E-mail: adrian.lloyd{at}warwick.ac.uk.

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