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Kinetic characterization of lipid II-Ala:alanyl-tRNA ligase (MurN) from Streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates.

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.

SUBMITTER: De Pascale G

PROVIDER: S-EPMC3259876 | biostudies-other | 2008 Dec

REPOSITORIES: biostudies-other

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Kinetic characterization of lipid II-Ala:alanyl-tRNA ligase (MurN) from Streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates.

De Pascale Gianfranco G Lloyd Adrian J AJ Schouten James A JA Gilbey Andrea M AM Roper David I DI Dowson Christopher G CG Bugg Timothy D H TD

The Journal of biological chemistry 20081008 50

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 ...[more]

PMID: 18842590

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Project description:Aminoacyl-tRNA synthetases provide the first step in protein synthesis quality control by discriminating cognate from noncognate amino acid and tRNA substrates. While substrate specificity is enhanced in many instances by cis- and trans-editing pathways, it has been revealed that in organisms such as Streptococcus pneumoniae some aminoacyl-tRNA synthetases display significant tRNA mischarging activity. To investigate the extent of tRNA mischarging in this pathogen, the aminoacylation profiles of class I isoleucyl-tRNA synthetase (IleRS) and class II lysyl-tRNA synthetase (LysRS) were determined. Pneumococcal IleRS mischarged tRNA(Ile) with both Val, as demonstrated in other bacteria, and Leu in a tRNA sequence-dependent manner. IleRS substrate specificity was achieved in an editing-independent manner, indicating that tRNA mischarging would only be significant under growth conditions where Ile is depleted. Pneumococcal LysRS was found to misaminoacylate tRNA(Lys) with Ala and to a lesser extent Thr and Ser, with mischarging efficiency modulated by the presence of an unusual U4:G69 wobble pair in the acceptor stems of both pneumococcal tRNA(Lys) isoacceptors. Addition of the trans-editing factor MurM, which also functions in peptidoglycan synthesis, reduced Ala-tRNA(Lys) production by LysRS, providing evidence for cross talk between the protein synthesis and cell wall biogenesis pathways. Mischarging of tRNA(Lys) by AlaRS was also observed, and this would provide additional potential MurM substrates. More broadly, the extensive mischarging activities now described for a number of Streptococcus pneumoniae aminoacyl-tRNA synthetases suggest that adaptive misaminoacylation may contribute significantly to the viability of this pathogen during amino acid starvation.Streptococcus pneumoniae is a common causative agent of several debilitating and potentially life-threatening infections, such as pneumonia, meningitis, and infectious endocarditis. Such infections are increasingly difficult to treat due to widespread development of penicillin resistance. High-level penicillin resistance is known to depend in part upon MurM, a protein involved in both aminoacyl-tRNA-dependent synthesis of indirect amino acid cross-linkages within cell wall peptidoglycan and in translation quality control. The involvement of MurM in both protein synthesis and antibiotic resistance identify it as a potential target for the development of new and potent antibiotics for pneumococcal infections. The goals of this work were to identify and characterize S. pneumoniae pathways that can synthesize mischarged tRNAs and to relate these activities to expected changes in protein and peptidoglycan biosynthesis during antibiotic and nutritional stress.

Dataset Information

Kinetic characterization of lipid II-Ala:alanyl-tRNA ligase (MurN) from Streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates.

Publications

Kinetic characterization of lipid II-Ala:alanyl-tRNA ligase (MurN) from Streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates.

Similar Datasets

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