Kinetic characterization of lipid II-Ala:alanyl-tRNA ligase (MurN) from Streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates.
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.
De Pascale
Gianfranco
G
Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, United Kingdom.
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
D000409:Alanine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004352:Drug Resistance, Microbial; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D010406:Penicillins / Q000737:chemistry; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013296:Streptococcus pneumoniae / Q000201:enzymology*
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D008055:Lipids; D010406:Penicillins; D011994:Recombinant Proteins; D012694:Serine; C408067:MurN protein, Streptococcus pneumoniae; D010453:Peptide Synthases; D000409:Alanine