Characterization of tRNA-dependent peptide bond formation by MurM in the synthesis of Streptococcus pneumoniae peptidoglycan

Abstract

MurMis 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 ε-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 MurMPn16 activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurMPn16 acylation activities were far lower than the alanylation activity of MurM 159. The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM159 compared with MurMPn16 bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA Ala:tRNASer 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 ε-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 TΨC loop stem of the tRNAAla. 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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.