Abstract
MurM and MurN are tRNA-dependent ligases that catalyze the addition of the first (L-Ala/L-Ser) and second (L-Ala) amino acid onto lipid II substrates in the biosynthesis of the peptidoglycan layer of Streptococcus pneumoniae. We have previously characterized the first ligase, MurM (Lloyd, A. J., Gilbey, A. M., Blewett, A. M., De Pascale, G., El Zoeiby, A., Levesque, R. C., Catherwood, A. C., Tomasz, A., Bugg, T. D., Roper, D. I., and Dowson, C. G. (2008) J. Biol. Chem. 283, 6402-6417). In order to characterize the second ligase MurN, we have developed a chemoenzymatic route to prepare the lipid II-Ala and lipid II-Ser substrates. Recombinant MurN enzymes from penicillin-resistant (159) and -sensitive (Pn16) S. pneumoniae were expressed and purified as MBP fusion proteins and reconstituted using a radiochemical assay. MurN ligases from strains 159 and Pn16 both showed a 20-fold higher catalytic efficiency for lipid II-L-Ala over lipid II-l-Ser, with no activity against unmodified lipid II, and similar kinetic parameters were measured for MurN from penicillin-resistant and penicillin-sensitive strains. These results concur with the peptidoglycan analysis of S. pneumoniae, in which the major cross-link observed is L-Ala-L-Ala. The combined action of ligases MurM and MurN is therefore required in order to rationalize the high level of dipeptide cross-links in penicillin-resistant S. pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains.
Highlights
Between L-lysine and D-alanine, but many Gram-positive bacteria contain a further peptide cross-link comprising one or more amino acids [3, 6]
The markedly different branching phenotype displayed by S. pneumoniae Pn16 and 159 is rationalized in vitro by the much higher specific activity of MurM159 over MurMPn16 with pneumococcal alanyl-tRNAAla and the higher activity with alanyltRNAAla than with seryl-tRNASer [1]
Overexpression, and Purification of MurN from S. pneumoniae—The murN genes from S. pneumoniae Pn16 and 159 strains were cloned into the expression vector pBADM-41 to allow expression of MurN fused to a maltose-binding protein (MBP)2 with N-terminal hexahistidine tag and C-terminal tobacco etch virus (TEV) protease cleavage site [27]
Summary
Staphylococcus aureus S. pneumoniae W. viridescens Enterococcus faecalis Enterococcus faecium Streptomyces coelicolor. Gly-Gly-Gly-Gly-Gly L-Ala-L-Ala or L-Ser-L-Ala L-Ala-L-Ser L-Ala-L-Ala D-Asxa Gly a D-Aspartate or D-asparagine. The MurN-catalyzed reaction, we have developed a chemoenzymatic method to prepare the aminoacyl-lipid II substrate for MurN, and we report the specificity of recombinant MurN for lipid II-Ala versus lipid II-Ser substrates
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