De Pascale, Gianfranco, Lloyd, Adrian J., Schouten, James A., Gilbey, Andrea M., Roper, David I., Dowson, Christopher G. and Bugg, Tim. (2008) Kinetic characterization of lipid II-Ala:Alanyl-tRNA ligase (MurN) from streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates. Journal of Biological Chemistry, Vol.283 (No.50). pp. 34571-34579. ISSN 0021-9258

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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 Mur M 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.

Item Type:	Journal Article
Subjects:	Q Science > QD Chemistry Q Science > QP Physiology Q Science > QR Microbiology
Divisions:	Faculty of Science > Chemistry Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Streptococcus pneumoniae, Peptidoglycans -- Synthesis, Ligases, Aminoacyl-tRNA synthetases
Journal or Publication Title:	Journal of Biological Chemistry
Publisher:	American Society for Biochemistry and Molecular Biology
ISSN:	0021-9258
Date:	12 December 2008
Volume:	Vol.283
Number:	No.50
Number of Pages:	9
Page Range:	pp. 34571-34579
Identification Number:	10.1074/jbc.M805807200
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Medical Research Council (Great Britain) (MRC), Wellcome Trust (London, England), Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC), University of Warwick, Sixth Framework Programme (European Commission) (FP6)
Grant number:	G0400848 (MRC), 066443 (WT)
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URI:	http://wrap.warwick.ac.uk/id/eprint/28834

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