Abstract
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, MurM(159) alanylated the peptidoglycan epsilon-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 MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) 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 MurM(159) 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 epsilon-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 TPsiC 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 beta-lactams for treatment of pneumococcal infection.
Highlights
The peptidoglycan in Streptococcus pneumoniae and other Gram-positive pathogens is composed of a carbohydrate polymer consisting of alternating residues of N-acetylglucosamine and N-acetylmuramic acid
The stem peptide is constructed in the cytoplasm appended to a UDP nucleotide (Fig. 1) in a series of reactions catalyzed by MurA to F, where MurC, -D, -E, and -F are responsible for the ATP-dependent ligation of L-alanine, D-glutamate, L-lysine, and D-alanyl-D-alanine, respectively, to form UDP-N-acetylmuramyl-L-alanyl-␥-D-glutamyl-L-lysyl-D-alanyl-D-alanine (UDP-MurNAcAEKAA) (1)
The phospho-Nacetylmuramyl pentapeptide is transferred from this species by MraY to a membrane-bound undecaprenyl-phosphate carrier to form lipid I, which is glycosylated with UDP-N-acetylglucosamine by MurG to form lipid II (undecaprenyl-pyrophosphoryl-Nacetylmuramyl (N-acetylglucosaminyl)-L-alanyl-␥-D-glutamylL-lysyl-D-alanyl-D-alanine) (1) (Fig. 1)
Summary
The peptidoglycan in Streptococcus pneumoniae and other Gram-positive pathogens is composed of a carbohydrate polymer consisting of alternating residues of N-acetylglucosamine and N-acetylmuramic acid. The pneumococcal stem peptide is further modified in S. pneumoniae where the lysyl residue ⑀-amino group is substituted by a dipeptide branch consisting of L-alanine or L-serine followed invariably by L-alanine (2– 4). After transport to the outer face of the cytoplasmic membrane, lipid II is polymerized by transglycosylation This nascent peptidoglycan is given structural rigidity by transpeptidation between the position 3 lysine (with or without a dipeptide branch) and the fourth position D-alanine of adjacent stem peptides (Fig. 1) (1). The pneumococcal genes encoding the enzymes that construct the dipeptide branch, MurM and MurN, add the first and second amino acids to the stem peptide lysine, respectively (10, 11). Extracellular Space to create a family of mosaics of related murM genes (12, 13) This has endowed the resulting family of MurM variants with vastly differing levels of activity in vivo and differing amino acid specificities for incorporation of alanine and serine (2, 12, 14). Recent successes in the in vitro synthesis of these precursors in our laboratory and elsewhere have, made the analysis of MurM enzymology a realistic proposition (17–19)
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